High arachidonic acid producing strains of yarrowia lipolytica

ABSTRACT

Engineered strains of the oleaginous yeast  Yarrowia lipolytica  capable of producing greater than 10% arachidonic acid (ARA, an ω-6 polyunsaturated fatty acid) in the total oil fraction are described. These strains comprise various chimeric genes expressing heterologous desaturases, elongases and acyltransferases, and optionally comprise various native desaturase and acyltransferase knockouts to enable synthesis and high accumulation of ARA. Production host cells are claimed, as are methods for producing ARA within said host cells.

This application claims the benefit of U.S. Provisional Application No.60/624,812, filed Nov. 4, 2004.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to an engineered strain of the oleaginous yeastYarrowia lipolytica that is capable of efficiently producing arachidonicacid (an ω-6 polyunsaturated fatty acid) in high concentrations.

BACKGROUND OF THE INVENTION

Arachidonic acid (ARA; cis-5,8,11,14-eicosatetraenoic; ω-6) is animportant precursor in the production of eicosanoids (e.g.,prostaglandins, thromboxanes, prostacyclin and leukot). Additionally,ARA is recognized as: (1) an essential long-chain polyunsaturated fattyacid (PUFA); (2) the principal ω-6 fatty acid found in the human brain;and, (3) an important component of breast milk and many infant formulas,based on its role in early neurological and visual development. Althoughadults obtain ARA readily from the diet in foods such as meat, eggs andmilk (and can also inefficiently synthesize ARA from dietary linolenicacid (LA)), commercial sources of ARA oil are typically produced fromnatural vegetarian sources (e.g., microorganisms in the generaMortierella (filamentous fungus), Entomophthora, Pythium andPorphyridium (red alga)). Most notably, Martek Biosciences Corporation(Columbia, Md.) produces an ARA-containing fungal oil (ARASCO®; U.S.Pat. No. 5,658,767) which is substantially free of EPA and which isderived from either Mortierella alpina or Pythium insidiuosum. One ofthe primary markets for this oil is infant formula; e.g., formulascontaining Martek's ARA oils are now available in more than 60 countriesworldwide.

Despite the availability of ARA from natural microbial sources such asthose described above, microbial production of ARA using recombinantmeans is expected to have several advantages over production fromnatural microbial sources. For example, recombinant microbes havingpreferred characteristics for oil production can be used, since thenaturally occurring microbial fatty acid profile of the host can bealtered by the introduction of new biosynthetic pathways in the hostand/or by the suppression of undesired pathways, thereby resulting inincreased levels of production of desired PUFAs (or conjugated formsthereof) and decreased production of undesired PUFAs. Secondly,recombinant microbes can provide PUFAs in particular forms which mayhave specific uses. And, finally, microbial oil production can bemanipulated by controlling culture conditions, notably by providingparticular substrate sources for microbially expressed enzymes, or byaddition of compounds/genetic engineering to suppress undesiredbiochemical pathways. Thus, for example, it is possible to modify theratio of ω-3 to ω-6 fatty acids so produced, or engineer production of aspecific PUFA (e.g., ARA) without significant accumulation of other PUFAdownstream or upstream products. The latter possibility is of particularinterest in some embodiments of the invention herein, wherein it isdesirable to provide a recombinant source of microbial oil containinghigh concentrations of ARA and that is additionally devoid ofgamma-linolenic acid (GLA; γ-linolenic acid; cis-6,9,12-octadecatrienoicacid; ω-6).

GLA is an important intermediate in the biosynthesis of biologicallyactive prostaglandin from LA. Although also recognized as an essentialω-6 PUFA having tremendous clinical, physiological and pharmaceuticalvalue, there are some applications in which GLA acts in opposition toARA. Thus, commercial production of an oil comprising ARA and devoid ofGLA would have utility in some applications.

Most microbially produced ARA is synthesized via the Δ6 desaturase/Δ6elongase pathway (which is predominantly found in, algae, mosses, fungi,nematodes and humans) and wherein: 1.) oleic acid is converted to LA bythe action of a Δ12 desaturase; 2.) LA is converted to GLA by the actionof a Δ6 desaturase; 3.) GLA is converted to DGLA by the action of aC_(18/20) elongase; and 3.) DGLA is converted to ARA by the action of aΔ5 desaturase (FIG. 1). However, an alternate Δ9 elongase/Δ8 desaturasepathway for the biosynthesis of ARA operates in some organisms, such aseuglenoid species, where it is the dominant pathway for formation of C₂₀PUFAs (Wallis, J. G., and Browse, J. Arch. Biochem. Biophys. 365:307-316(1999); WO 00/34439; and Qi, B. et al. FEBS Letters. 510:159-165(2002)). In this pathway, LA is converted to EDA by a Δ9 elongase, EDAis converted to DGLA by a Δ8 desaturase, and DGLA is converted to ARA bya Δ5 desaturase.

Although genes encoding the Δ6 desaturase/Δ6 elongase and the Δ9elongase/Δ8 desaturase pathways have now been identified andcharacterized from a variety of organisms, and some have beenheterologously expressed in combination with other PUFA desaturases andelongases, neither of these pathways have been introduced into amicrobe, such as a yeast, and manipulated via complex metabolicengineering to enable economical production of commercial quantities ofARA (i.e., greater than 10% with respect to total fatty acids).Additionally, considerable discrepancy exists concerning the mostappropriate choice of host organism for such engineering.

Recently, Picataggio et al. (WO 2004/101757 and co-pending U.S. PatentApplication No. 60/624,812) have explored the utility of oleaginousyeast, and specifically, Yarrowia lipolytica (formerly classified asCandida lipolytica), as a preferred class of microorganisms forproduction of PUFAs such as ARA and EPA. Oleaginous yeast are defined asthose yeast that are naturally capable of oil synthesis andaccumulation, wherein oil accumulation can be up to about 80% of thecellular dry weight. Despite a natural deficiency in the production ofω-6 and ω-3 fatty acids in these organisms (since naturally producedPUFAs are limited to 18:2 fatty acids (and less commonly, 18:3 fattyacids)), Picataggio et al. (supra) have demonstrated production of 1.3%ARA and 1.9% EPA (of total fatty acids) in Y. lipolytica usingrelatively simple genetic engineering approaches and up to 28% EPA usingmore complex metabolic engineering. However, similar work has not beenperformed to enable economic, commercial production of ARA in thisparticular host organism.

Applicants have solved the stated problem by engineering various strainsof Yarrowia lipolytica that are capable of producing greater than 10-14%ARA in the total oil fraction, using either the Δ6 desaturase/Δ6elongase pathway or the Δ9 elongase/Δ8 desaturase pathway (therebyproducing 10-11% ARA-oil with 25-29% GLA or 14% ARA-oil that is devoidof GLA, respectively). Additional metabolic engineering and fermentationmethods are provided to further enhance ARA productivity in thisoleaginous yeast.

SUMMARY OF THE INVENTION

The invention relates to recombinant production hosts of the genusYarrowia having enzymatic pathways useful of the production ofarachidonic acid.

Accordingly the invention provides a recombinant production host cellfor the production of arachidonic acid comprising a background Yarrowiasp. comprising a gene pool comprising the following genes of the ω-3/ω-6fatty acid biosynthetic pathway:

-   -   a) at least one gene encoding Δ6 desaturase;    -   b) at least one gene encoding C_(18/20) elongase; and,    -   c) at least one gene encoding Δ5 desaturase;        wherein at least one of said ω-3/ω-6 fatty acid biosynthetic        pathway genes is over-expressed.

In another embodiment the invention provides a recombinant productionhost cell for the production of arachidonic acid comprising a backgroundYarrowia sp. comprising a gene pool comprising the following genes ofthe ω-3/ω-6 fatty acid biosynthetic pathway:

-   -   a) at least one gene encoding Δ9 elongase;    -   b) at least one gene encoding Δ8 desaturase; and,    -   c) at least one gene encoding Δ5 desaturase;        wherein at least one of said ω-3/ω-6 fatty acid biosynthetic        pathway genes is over-expressed.

In specific embodiments recombinant production hosts of the inventionmay additionally comprise additional pathway elements including, but notlimited to: at least one gene encoding Δ12 desaturase; at least one geneencoding Δ9 desaturase; at least one gene encoding C_(16/18) elongase;and at least one gene encoding C_(14/16) elongase and at least one geneencoding an acyltransferase.

In one specific embodiment the invention provides a recombinantproduction host cell for the production of arachidonic acid comprising abackground Yarrowia sp. comprising a gene pool comprising the followinggenes of the ω-3/ω-6 fatty acid biosynthetic pathway:

-   -   a) at least one gene encoding Δ6 desaturase; and,    -   b) at least one gene encoding C_(18/20) elongase; and,    -   c) at least one gene encoding Δ5 desaturase; and,    -   d) at least one gene encoding Δ12 desaturase;

wherein the background Yarrowia sp. is devoid of any native geneencoding an isopropyl malate dehydrogenase (Leu2−) enzyme; and,

wherein at least one of said ω-3/ω-6 fatty acid biosynthetic pathwaygenes is over-expressed.

In another specific embodiment the invention provides a recombinantproduction host cell for the production of arachidonic acid comprising abackground Yarrowia sp. comprising a gene pool comprising the followinggenes of the ω-3/ω-6 fatty acid biosynthetic pathway:

-   -   a) at least one gene encoding Δ9 elongase; and,    -   b) at least one gene encoding Δ8 desaturase; and,    -   c) at least one gene encoding Δ5 desaturase; and,    -   d) at least one gene encoding Δ12 desaturase; and,    -   e) at least one gene encoding C_(16/18) elongase;

wherein the background Yarrowia sp. is devoid of any native geneencoding a saccharopine dehydrogenase (Lys5−) enzyme; and,

wherein at least one of said ω-3/ω-6 fatty acid biosynthetic pathwaygenes is over-expressed.

In another embodiment the invention provides a method for the productionof a microbial oil comprising arachidonic acid comprising:

-   -   a) culturing the production host of any of claim 1, or 2 wherein        a microbial oil comprising arachidonic acid is produced; and    -   b) optionally recovering the microbial oil of step (a).

In another embodiment the invention provides a microbial oil produced bythe methods of the invention and using the recombinant production hostsof the invention.

In an alternate embodiment the invention provides a food productcomprising an effective amount of a microbial oil produced by the methodof the invention.

In a specific embodiment the invention provides product selected fromthe group consisting of a medical food, a dietary supplement; infantformula and a pharmaceutical comprising an effective amount of amicrobial oil produced by the method of the invention.

In an alternate embodiment the invention provides an animal feedcomprising an effective amount of the microbial oil produced by themethod of the invention.

The invention additionally provides methods of making a product a foodproduct, or an animal feed supplemented with arachidonic acid comprisingcombining a microbial oil produced by the methods of the invention withproduct, food product or animal feed.

In another embodiment the invention provides a method for providing ahuman, animal or aquaculture organism diet supplement enriched witharachidonic acid (ARA) comprising providing a microbial oil produced bythe method of the invention containing arachidonic acid in a formconsumable or usable by humans or animals.

Biological Deposits

The following biological materials have been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, and bear the following designations, accession numbersand dates of deposit.

Biological Material Accession Number Date of Deposit Plasmid pY89-5 ATCCPTA-6048 Jun. 4^(th), 2004 Yarrowia lipolytica Y2047 ATCC PTA-

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1 illustrates the ω-3/ω-6 fatty acid biosynthetic pathway.

FIG. 2 is a schematic illustration of the biochemical mechanism forlipid accumulation in oleaginous yeast.

FIG. 3 is a schematic illustration describing the role of variousacyltransferases in lipid accumulation in oleaginous yeast.

FIG. 4 diagrams the development of some Yarrowia lipolytica strains ofthe invention, producing various fatty acids (including ARA) in thetotal lipid fraction.

FIG. 5A provides a plasmid map for pY5-30. FIG. 5B illustrates therelative promoter activities of TEF, GPD, GPM, FBA and FBAIN in Yarrowialipolytica ATCC #76982 strains, as determined by histochemical staining.FIG. 5C illustrates the relative promoter activities of YAT1, TEF, GPATand FBAIN in Y. lipolytica grown in various media as determined byhistochemical staining.

FIG. 6A is a graph comparing the promoter activity of GPD, GPM, FBA andFBAIN in Yarrowia lipolytica ATCC #76982 strains, as determinedfluorometrically. FIG. 6B graphically summarizes the results of RealTime PCR relative quanitation, wherein the GUS mRNA in Y. lipolyticaATCC #76982 strains (i.e., expressing GPD::GUS, GPDIN::GUS, FBA::GUS orFBAIN::GUS chimeric genes) was quantified to the mRNA level of the Y.lipolytica strain expressing pY5-30 (i.e., a chimeric TEF::GUS gene).

FIG. 7 provides plasmid maps for the following: (A) pY57.YI.AHAS.w4971;(B) pKUNF12T6E; (C) pDMW232; and (D) pDMW271.

FIG. 8 provides plasmid maps for the following: (A) pKUNT2; (B) pZUF17;(C) pDMW237; (D) pDMW240; and (E) yeast expression vector pY89-5.

FIG. 9 shows a chromatogram of the lipid profile of an Euglena graciliscell extract.

FIG. 10 shows an alignment of various Euglena gracilis Δ8 desaturasepolypeptide sequences. The method of alignment used corresponds to the“Clustal V method of alignment”.

FIG. 11 provides plasmid maps for the following: (A) pKUNFmKF2; (B)pDMW277; (C) pZF5T-PPC; (D) pDMW287F; and (E) pDMW297.

FIG. 12 provides plasmid maps for the following: (A) pZP2C16M899; (B)pDMW314; (C) pDM322; and (D) pZKL5598.

FIG. 13 provides plasmid maps for the following: (A) pZP3L37; (B)pY37/F15; (C) pKO2UF2PE; and (D) pZKUT16.

FIG. 14 provides plasmid maps for the following: (A) pKO2UM25E; (B)pZKUGPI5S; (C) pDMW302T16; and (D) pZKUGPE1S.

FIG. 15 provides plasmid maps for the following: (A) pKO2UM26E; (B)pZUF-Mod-1; (C) pMDAGAT1-17; and (D) pMGPAT-17.

FIG. 16 graphically represents the relationship between SEQ ID NOs:97,98, 99, 100, 101, 102, 103, 104, 105, 106 and 107, each of which relatesto glycerol-3-phosphate o-acyltransferase (GPAT) in Mortierella alpina.

FIG. 17 graphically represents the relationship between SEQ ID NOs:53,54, 55, 56, 57, 58, 59 and 60, each of which relates to the C_(16/18)fatty acid elongase enzyme (ELO3) in Mortierella alpina.

FIG. 18 provides plasmid maps for the following: (A) pZUF6S; (B)pZUF6S-E3WT; (C) pZKUGPYE1-N; and (D) pZKUGPYE2.

FIG. 19 provides plasmid maps for the following: (A) pZKUGPYE1; (B)pZUF6FYE1; (C) pZP2I7+Ura; (D) pY20; and (E) pLV13.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1-112, 158-168, 209, 252, 255 and 357-364 are ORFs encodingpromoters, genes or proteins (or fragments thereof) as identified inTable 1.

TABLE 1 Summary of Gene and Protein SEQ ID Numbers Nucleic acid ProteinDescription SEQ ID NO. SEQ ID NO. Mortierella alpina Δ6 desaturase 1(1374 bp) 2 (457 AA) Synthetic A6 desaturase, derived from 3 (1374 bp) 2(457 AA) Mortierella alpina, codon-optimized for expression in Yarrowialipolytica Mortierella alpina Δ6 desaturase “B” 4 (1521 bp) 5 (458 AA)Mortierella alpina Δ5 desaturase 6 (1341 bp) 7 (446 AA) Isochrysisgalbana Δ5 desaturase 8 (1329 bp) 9 (442 AA) Synthetic Δ5 desaturasederived from 10 (1329 bp) 9 (442 AA) Isochrysis galbana, codon-optimizedfor expression in Yarrowia lipolytica Homo sapiens Δ5 desaturase 11(1335 bp) 12 (444 AA) Synthetic Δ5 desaturase derived from 13 (1335 bp)12 (444 AA) Homo sapiens, codon-optimized for expression in Yarrowialipolytica Saprolegnia diclina Δ17 desaturase 14 (1077 bp) 15 (358 AA)Synthetic Δ17 desaturase gene derived 16 (1077 bp) 15 (358 AA) fromSaprolegnia diclina, codon-optimized for expression in Yarrowialipolytica Mortierella alpina C_(18/20) elongase 17 (957 bp) 18 (318 AA)Synthetic C_(18/20) elongase gene derived 19 (957 bp) 18 (318 AA) fromMortierella alpina, codon-optimized for expression in Yarrowialipolytica Thraustochytrium aureum C_(18/20) elongase 20 (819 bp) 21(272 AA) Synthetic C_(18/20) elongase gene derived 22 (819 bp) 21 (272AA) from Thraustochytrium aureum, codon- optimized for expression inYarrowia lipolytica Yarrowia lipolytica Δ12 desaturase 23 (1936 bp) 24(419 AA) Mortieralla isabellina Δ12 desaturase 25 (1203 bp) 26 (400 AA)Fusarium moniliforme Δ12 desaturase 27 (1434 bp) 28 (477 AA) Aspergillusnidulans Δ12 desaturase 29 (1416 bp) 30 (471 AA) Aspergillus flavus Δ12desaturase — 31 (466 AA) Aspergillus fumigatus Δ12 desaturase — 32 (424AA) Magnaporthe grisea Δ12 desaturase 33 (1656 bp) 34 (551 AA)Neurospora crassa Δ12 desaturase 35 (1446 bp) 36 (481 AA) Fusariumgraminearium Δ12 desaturase 37 (1371 bp) 38 (456 AA) Mortierella alpinaΔ12 desaturase 357 (1403 bp) 358 (400 AA) Saccharomyces kluyveri Δ12desaturase — 359 (416 AA) Kluyveromyces lactis Δ12 desaturase 360 (1948bp) 361 (415 AA) Candida albicans Δ12 desaturase — 362 (436 AA)Debaryomyces hansenii CBS767 Δ12 — 363 (416 AA) desaturase Isochrysisgalbana Δ9 elongase 39 (792 bp) 40 (263 AA) Synthetic Δ9 elongase gene,codon- 41 (792 bp) 40 (263 AA) optimized for expression in Yarrowialipolytica Euglena gracillis Δ8 desaturase gene 42 (1275 bp) 43 (419 AA)(non-functional; GenBank Accession No. AAD45877) Euglena gracillis Δ8desaturase gene — 252 (422 AA) (non-functional; Wallis et al. [Archivesof Biochem. Biophys., 365: 307-316 (1999)]; WO 00/34439) Synthetic Δ8desaturase gene, codon- 209 (1270 bp) — optimized for expression inYarrowia lipolytica (D8S-1) Synthetic Δ8 desaturase gene, codon- 255(1269 bp) — optimized for expression in Yarrowia lipolytica (D8S-3)Euglena gracillis Δ8 desaturase gene 44 (1271 bp) 45 (421 AA) (Eg5)Euglena gracillis Δ8 desaturase gene 46 (1271 bp) 47 (421 AA) (Eg12)Synthetic Δ8 desaturase gene, codon- 48 (1272 bp) 49 (422 AA) optimizedfor expression in Yarrowia lipolytica (D8SF) Rattus norvegicus C_(16/18)elongase 50 (2628 bp) 51 (267 AA) Synthetic C_(16/18) elongase genederived 52 (804 bp) 51 (267 AA) from Rattus norvegicus, codon-optimizedfor expression in Yarrowia lipolytica Mortierella alpina C_(16/18)elongase (ELO3) 53 (828 bp) 54 (275 AA) Mortierella alpina ELO3—partialcDNA 55 (607 bp) — sequence Mortierella alpina ELO3—3′ sequence 56 (1042bp) — obtained by genome walking Mortierella alpina ELO3—5′ sequence 57(2223 bp) — obtained by genome walking Mortierella alpina ELO3—cDNAcontig 58 (3557 bp) — Mortierella alpina ELO3—intron 59 (542 bp) —Mortierella alpina ELO3—genomic contig 60 (4099 bp) — Yarrowialipolytica C_(16/18) elongase gene 61 (915 bp) 62 (304 AA) Candidaalbicans probable fatty acid — 63 (353 AA) elongase (GenBank AccessionNo. EAL04510) Yarrowia lipolytica C_(14/16) elongase gene 64 (978 bp) 65(325 AA) Neurospora crassa FEN1 gene (GenBank — 66 (337 AA) AccessionNo. CAD70918) Mortierella alpina lysophosphatidic acid 67 (945 bp) 68(314 AA) acyltransferase (LPAAT1) Mortierella alpina lysophosphatidicacid 69 (927 bp) 70 (308 AA) acyltransferase (LPAAT2) Yarrowialipolytica lysophosphatidic acid 71 (1549 bp) 72 (282 AA)acyltransferase (LPAAT1) Yarrowia lipolytica lysophosphatidic acid 73(1495 bp) — acyltransferase (LPAAT2)—genomic fragment comprising geneYarrowia lipolytica lysophosphatidic acid 74 (672 bp) 75 (223 AA)acyltransferase (LPAAT2) Yarrowia lipolytica 76 (2326 bp) 77 (648 AA)phospholipid:diacylglycerol acyltransferase (PDAT) Yarrowia lipolyticaacyl-CoA:sterol- 78 (1632 bp) 79 (543 AA) acyltransferase (ARE2)Caenorhabditis elegans acyl-CoA:1-acyl — 80 (282 AA)lysophosphatidylcholine acyltransferase (LPCAT) Yarrowia lipolyticadiacylglycerol 81 (1578 bp) 82 (526 AA) acyltransferase (DGAT1)Mortierella alpina diacylglycerol 83 (1578 bp) 84 (525 AA)acyltransferase (DGAT1) Neurospora crassa diacylglycerol — 85 (533 AA)acyltransferase (DGAT1) Gibberella zeae PH-1 diacylglycerol — 86 (499AA) acyltransferase (DGAT1) Magnaporthe grisea diacylglycerol — 87 (503AA) acyltransferase (DGAT1) Aspergillus nidulans diacylglycerol — 88(458 AA) acyltransferase (DGAT1) Yarrowia lipolytica diacylglycerol 89(2119 bp) 90 (514 AA) acyltransferase (DGAT2) 91 (1380 bp) 92 (459 AA)93 (1068 bp) 94 (355 AA) Mortierella alpina diacylglycerol 95 (996 bp)96 (331 AA) acyltransferase (DGAT2) Mortierella alpinaglycerol-3-phosphate 97 (2151 bp) 98 (716 AA) acyltransferase (GPAT) M.alpina GPAT—partial cDNA sequence 99 (1212 bp) — M. alpina GPAT—genomicfragment 100 (3935 bp) — comprising −1050 bp to +2886 bp region M.alpina GPAT—3′ cDNA sequence 101 (965 bp) — obtained by genome walkingM. alpina GPAT—5′ sequence obtained 102 (1908 bp) — by genome walking M.alpina GPAT—internal sequence 103 (966 bp) — obtained by genome walkingM. alpina GPAT—intron #1 104 (275 bp) — M. alpina GPAT—intron #2 105(255 bp) — M. alpina GPAT—intron #3 106 (83 bp) — M. alpina GPAT—intron#4 107 (99 bp) — Yarrowia lipolytica diacylglycerol 108 (2133 bp) —cholinephosphotransferase (CPT1)—genomic fragment comprising geneYarrowia lipolytica diacylglycerol 109 (1185 bp) 110 (394 AA)cholinephosphotransferase (CPT1) Saccharomyces cerevisiae inositol 111(1434 bp) 112 (477 AA) phosphosphingolipid-specific phospholipase C(ISC1) Yarrowia lipolytica glyceraldehyde-3- 158 (971 bp) — phosphatedehydrogenase promoter (GPD) Yarrowia lipolytica glyceraldehyde-3- 159(1174 bp) — phosphate dehydrogenase + intron promoter (GPDIN) Yarrowialipolytica phosphoglycerate 160 (878 bp) — mutase promoter (GPM)Yarrowia lipolytica fructose-bisphosphate 161 (1001 bp) — aldolasepromoter (FBA) Yarrowia lipolytica fructose-bisphosphate 162 (973 bp) —aldolase + intron promoter (FBAIN) Yarrowia lipolyticafructose-bisphosphate 163 (924 bp) — aldolase + modified intron promoter(FBAINm) Yarrowia lipolytica glycerol-3-phosphate 164 (1130 bp) —acyltransferase promoter (GPAT) Yarrowia lipolytica ammonium transporter165 (778 bp) — promoter (YAT1) Yarrowia lipolytica translationelongation 166 (436 bp) — factor EF1-α promoter (TEF) Yarrowialipolytica chimeric GPM::FBA 167 (1020 bp) — intron promoter(GPM::FBAIN) Yarrowia lipolytica chimeric GPM::GPD 168 (1052 bp) —intron promoter (GPM::GPDIN) Yarrowia lipolytica export protein 364(1000 bp) — promoter (EXP1)

SEQ ID NOs:113-157 are plasmids as identified in Table 2.

TABLE 2 Summary of Plasmid SEQ ID Numbers Plasmid Corresponding FigureSEQ ID NO pY5-30  5A 113 (8,953 bp) pKUNF12T6E  7B 114 (12,649 bp)pDMW232  7C 115 (10,945 bp) pDMW271  7D 116 (13,034 bp) pKUNT2  8A 117(6,457 bp) pZUF17  8B 118 (8,165 bp) pDMW237  8C 119 (7,879 bp) pY54PC —120 (8,502 bp) pKUNFmkF2 11A 121 (7,145 bp) pZF5T-PPC 11C 122 (5,553 bp)pDMW297 11E 123 (10,448 bp) pZP2C16M899 12A 124 (15,543 bp) pDMW314 12B125 (13,295 bp) pDMW322 12C 126 (11,435 bp) pZKSL5598 12D 127 (16,325bp) pZP3L37 13A 128 (12,690 bp) pY37/F15 13B 129 (8,194 bp) pKO2UF2PE13C 130 (10,838 bp) pZKUT16 13D 131 (5,833 bp) pKO2UM25E 14A 132 (12,663bp) pZKUGPI5S 14B 133 (6,912 bp) pDMW302T16 14C 134 (14,864 bp)pZKUGPE1S 14D 135 (6,540 bp) pKO2UM26E 15A 136 (13,321 bp) pZKUM — 137(4,313 bp) pMLPAT-17 — 138 (8,015 bp) pMLPAT-Int — 139 (8,411 bp)pZUF-MOD-1 15B 140 (7,323 bp) pMDGAT1-17 15C 141 (8,666 bp) pMDGAT2-17 —142 (8,084 bp) pMGPAT-17 15D 143 (9,239 bp) pZF5T-PPC-E3 — 144 (5,031bp) pZUF6S 18A 145 (8,462 bp) pZUF6S-E3WT 18B 146 (11,046 bp)pZKUGPYE1-N 18C 147 (6,561 bp) pZKUGPYE2 18D 148 (6,498 bp) pZUF6TYE2 —149 (10,195 bp) pZKUGPYE1 19A 150 (6,561 bp) pZUF6FYE1 19B 151 (10,809bp) pYCPT1-17 — 152 (8,273 bp) pZP2I7 + Ura 19C 153 (7,822 bp)pYCPT1-ZP2I7 — 154 (7,930 bp) pTEF::ISC1 — 155 (8,179 bp) pY20 19D 156(8,196 bp) pLV13 19E 157 (5,105 bp)

SEQ ID NO:356 corresponds to the codon-optimized translation initiationsite for genes optimally expressed in Yarrowia sp.

SEQ ID NOs:169-182 correspond to primers YL211, YL212, YL376, YL377,YL203, YL204, GPAT-5-1, GPAT-5-2, ODMW314, YL341, ODMW320, ODMW341,27203-F and 27203-R, respectively, used to amplify Yarrowia lipolyticapromoter regions.

SEQ ID NOs:183-186 are the oligonucleotides YL-URA-16F, YL-URA-78R,GUS-767F and GUS-891R, respectively, used for Real Time analysis.

SEQ ID NOs:187-202 correspond to 8 pairs of oligonucleotides whichtogether comprise the entire codon-optimized coding region of the I.galbana Δ9 elongase (i.e., IL3-1A, IL3-1B, IL3-2A, IL3-2B, IL3-3A,IL3-3B, IL3-4A, IL3-4B, IL3-5A, IL3-5B, IL3-6A, IL3-6B, IL3-7A, IL3-7B,IL3-8A and IL3-8B, respectively).

SEQ ID NOs:203-206 correspond to primers IL3-1F, IL3-4R, IL3-5F andIL3-8R, respectively, used for PCR amplification during synthesis of thecodon-optimized Δ9 elongase gene.

SEQ ID NO:207 is the 417 bp NcoI/PstI fragment described in pT9(1-4);and SEQ ID NO:208 is the 377 bp PstI/Not1 fragment described inpT9(5-8).

SEQ ID NOs:210-235 correspond to 13 pairs of oligonucleotides whichtogether comprise the entire codon-optimized coding region of the E.gracilis Δ8 desaturase (i.e., D8-1A, D8-1B, D8-2A, D8-2B, D8-3A, D8-3B,D8-4A, D8-4B, D8-5A, D8-5B, D8-6A, D8-6B, D8-7A, D8-7B, D8-8A, D8-8B,D8-9A, D8-9B, D8-10A, D8-10B, D8-11A, D8-11B, D8-12A, D8-12B, D8-13A andD8-13B, respectively).

SEQ ID NOs:236-243 correspond to primers D8-1F, D8-3R, D8-4F, D8-6R,D8-7F, D8-9R, D8-10F and D8-13R, respectively, used for PCRamplification during synthesis of the codon-optimized Δ8 desaturasegene.

SEQ ID NO:244 is the 309 bp Nco/BglII fragment described in pT8(1-3);SEQ ID NO:245 is the 321 bp BglII/XhoI fragment described in pT8(4-6);SEQ ID NO:246 is the 264 bp XhoI/SacI fragment described in pT8(7-9);and SEQ ID NO:247 is the 369 bp Sac1/Not1 fragment described inpT8(10-13).

SEQ ID NOs:248 and 249 correspond to primers ODMW390 and ODMW391,respectively, used during synthesis of D8S-2 in pDMW255.

SEQ ID NOs:250 and 251 are the chimeric D8s⁻¹::XPR and D8S-2::XPR genesdescribed in Example 7.

SEQ ID NOs:253 and 254 correspond to primers ODMW392 and ODMW393, usedduring synthesis of D8S-3.

SEQ ID NOs:256 and 257 correspond to primers Eg5-1 and Eg3-3,respectively, used for amplification of the Δ8 desaturase from Euglenagracilis.

SEQ ID NOs:258-261 correspond to primers T7, M13-28Rev, Eg3-2 and Eg5-2,respectively, used for sequencing a Δ8 desaturase clone.

SEQ ID NO:262 corresponds to primer ODMW404, used for amplification ofD8S-3.

SEQ ID NO:263 is a 1272 bp chimeric gene comprising D8S-3.

SEQ ID NOs:264 and 265 correspond to primers YL521 and YL522,respectively, used to create new restriction enzyme sites in a clonedD8S-3 gene.

SEQ ID NOs:266-279 correspond to primers YL525, YL526, YL527, YL528,YL529, YL530, YL531, YL532, YL533, YL534, YL535, YL536, YL537 and YL538,respectively, used in site directed mutagenesis reactions to produceD8SF.

SEQ ID NO:280 is a mutant AHAS gene comprising a W497L mutation.

SEQ ID NOs:281-283 correspond to BD-Clontech Creator Smart® cDNA librarykit primers SMART IV oligonucleotide, CDSIII/3′ PCR primer and 5′-PCRprimer, respectively.

SEQ ID NO:284 corresponds to the M13 forward primer used for M. alpinacDNA library sequencing.

SEQ ID NOs:285-288 and 290-291 correspond to primers MLPAT-F, MLPAT-R,LPAT-Re-5-1, LPAT-Re-5-2, LPAT-Re-3-1 and LPAT-Re-3-2, respectively,used for cloning of the M. alpina LPAAT2 ORF.

SEQ ID NOs:289 and 292 correspond to a 5′ (1129 bp) and 3′ (938 bp)region of the Y. lipolytica LPAAT1 ORF, respectively.

SEQ ID NOs:293 and 294 correspond to primers pzuf-mod 1 and pzuf-mod 2,respectively, used for creating “control” plasmid pZUF-MOD-1.

SEQ ID NOs:295 and 296 correspond to primers MACAT-F1 and MACAT-R,respectively, used for cloning of the M. alpina DGAT1 ORF.

SEQ ID NOs:297 and 298 correspond to primers MDGAT-F and MDGAT-R1,respectively, used for cloning of the M. alpina DGAT2 ORF.

SEQ ID NOs:299 and 300 correspond to primers MGPAT-N1 and MGPAT-NR5,respectively, used for degenerate PCR to amplify the M. alpina GPAT.

SEQ ID NOs:301-303 correspond to primers MGPAT-5N1, MGPAT-5N2 andMGPAT-5N3, respectively, used for amplification of the 3′-end of the M.alpina GPAT.

SEQ ID NOs:304 and 305 correspond to the Genome Walker adaptor fromClonTech's Universal GenomeWalker™ Kit, used for genome-walking.

SEQ ID NOs:306-309 correspond to the PCR primers used in genome-walking:MGPAT-5-1A, Adaptor-1 (AP1), MGPAT-3N1 and Nested Adaptor Primer 2(AP2), respectively.

SEQ ID NOs:310 and 311 correspond to primers mgpat-cdna-5 andmgpat-cdna-R, respectively, used for amplifying the M. alpina GPAT.

SEQ ID NOs:312 and 313 correspond to primers MA Elong 3′1 and MA elong3′2, respectively, used for genome-walking to isolate the 3′-end regionof the M. alpina ELO3.

SEQ ID NOs:314 and 315 correspond to primers MA Elong 5′1 and MA Elong5′2, respectively, used for genome-walking to isolate the 5′-end regionof the M. alpina ELO3.

SEQ ID NOs:316 and 317 correspond to primers MA ELONG 5′ NcoI 3 and MAELONG 3′ NotI 1, respectively, used for amplifying the complete ELO3from M. alpina cDNA.

SEQ ID NOs:318 and 319 correspond to primers YL597 and YL598,respectively, used for amplifying the coding region of Y. lipolyticaYE2.

SEQ ID NOs:320-323 correspond to primers YL567, YL568, YL569 and YL570,respectively, used for amplifying the coding region of Y. lipolyticaYE1.

SEQ ID NOs:324 and 325 correspond to primers YL571 and YL572,respectively, used for site-directed mutagenesis during cloning of Y.lipolytica YE1.

SEQ ID NOs:326 and 327 correspond to primers CPT1-5′-NcoI andCPT1-3′-Not/, respectively, used for cloning of the Y. lipolytica CPT1ORF.

SEQ ID NOs: 328 and 329 correspond to primers Isc1F and Isc1R,respectively, used for cloning of the S. cerevisiae ISC1 ORF.

SEQ ID NOs:330 and 331 correspond to primers Pcl1F and Pcl1R,respectively, used for cloning of the S. cerevisiae PCL1 ORF.

SEQ ID NOs:332-335 correspond to primers P95, P96, P97 and P98,respectively, used for targeted disruption of the Y. lipolytica DGAT2gene.

SEQ ID NOs:336-338 correspond to primers P115, P116 and P112,respectively, used to screen for targeted integration of the disruptedY. lipolytica DGAT2 gene.

SEQ ID NOs:339-342 correspond to primers P39, P41, P40 and P42,respectively, used for targeted disruption of the Y. lipolytica PDATgene.

SEQ ID NOs:343-346 correspond to primers P51, P52, P37 and P38,respectively, used to screen for targeted integration of the disruptedY. lipolytica PDAT gene.

SEQ ID NOs:347 and 348 are the degenerate primers identified as P201 andP203, respectively, used for the isolation of the Y. lipolytica DGAT1.

SEQ ID NOs:349-353 correspond to primers P214, P215, P216, P217 andP219, respectively, used for the creation of a targeting cassette fortargeted disruption of the putative DGAT1 gene in Y. lipolytica.

SEQ ID NOs:354 and 355 correspond to primers P226 and P227,respectively, used to screen for targeted integration of the disruptedY. lipolytica DGAT1 gene.

SEQ ID NOs:365-370 correspond to primers 410, 411, 412, 413, 414 and415, respectively, used for synthesis of a mutant Yarrowia lipolyticaAHAS gene, comprising a W497L mutation.

SEQ ID NOs:371 and 372 correspond to primers YL325 and YL326,respectively, used to amplify a NotI/PacI fragment containing the Aco 3′terminator.

SEQ ID NO:373 corresponds to a His Box 1 motif found in fungal Δ15 andΔ12 desaturases.

SEQ ID NO:374 corresponds to a motif that is indicative of a fungalprotein having Δ15 desaturase activity, while SEQ ID NO:375 correspondsto a motif that is indicative of a fungal protein having Δ12 desaturaseactivity.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and publications cited herein areincorporated by reference in their entirety. This specifically includes,the following Applicants' Assignee's copending applications:

-   U.S. patent application Ser. No. 10/840,478 (filed May 6, 2004),-   U.S. patent application Ser. No. 10/840,579 (filed May 6, 2004),-   U.S. patent application Ser. No. 10/840,325 (filed May 6, 2004)-   U.S. patent application Ser. No. 10/869,630 (filed Jun. 16, 2004),-   U.S. patent application Ser. No. 10/882,760 (filed Jul. 1, 2004),-   U.S. patent application Ser. No. 10/985,109 (filed Nov. 10, 2004),-   U.S. patent application Ser. No. 10/987,548 (filed Nov. 12, 2004)-   U.S. Patent Application No. 60/624,812 (filed Nov. 4, 2004),-   U.S. patent application Ser. No. 11/024,545 and Ser. No. 11/024,544    (filed Dec. 29, 2004),-   U.S. Patent Application No. 60/689,031 (filed Jun. 9, 2005),-   U.S. patent application Ser. No. 11/183,664 (filed Jul. 18, 2005),-   U.S. patent application Ser. No. 11/185,301 (filed Jul. 20, 2005),-   U.S. patent application Ser. No. 11/190,750 (filed Jul. 27, 2005),-   U.S. patent application Ser. No. 11/225,354 (filed Sep. 13, 2005),    CL2823 and CL3027

In accordance with the subject invention, Applicants provide productionhost strains of Yarrowia lipolytica that are capable of producinggreater than 10% arachidonic acid (ARA, 20:4, ω-6). Accumulation of thisparticular polyunsaturated fatty acid (PUFA) is accomplished byintroduction of either of two different functional ω-3/ω-6 fatty acidbiosynthetic pathways. The first pathway comprises proteins with Δ6desaturase, C_(18/20) elongase and Δ5 desaturase activities into theoleaginous yeast host for high-level recombinant expression, wherein theARA oil also comprises GLA; the latter pathway comprises proteins withΔ9 elongase, Δ8 desaturase and Δ5 desaturase activities and therebyenables production of an ARA oil that is devoid of any GLA. Thus, thisdisclosure demonstrates that Y. lipolytica can be engineered to enablecommercial production of ARA and derivatives thereof. Methods ofproduction are also claimed.

The subject invention finds many applications. PUFAs, or derivativesthereof, made by the methodology disclosed herein can be used as dietarysubstitutes, or supplements, particularly infant formulas, for patientsundergoing intravenous feeding or for preventing or treatingmalnutrition. Alternatively, the purified PUFAs (or derivatives thereof)may be incorporated into cooking oils, fats or margarines formulated sothat in normal use the recipient would receive the desired amount fordietary supplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements or other food products and may finduse as anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use (human or veterinary).In this case, the PUFAs are generally administered orally but can beadministered by any route by which they may be successfully absorbed,e.g., parenterally (e.g., subcutaneously, intramuscularly orintravenously), rectally, vaginally or topically (e.g., as a skinointment or lotion).

Supplementation of humans or animals with PUFAs produced by recombinantmeans can result in increased levels of the added PUFAs, as well astheir metabolic progeny. For example, treatment with ARA can result notonly in increased levels of ARA, but also downstream products of ARAsuch as eicosanoids. Complex regulatory mechanisms can make it desirableto combine various PUFAs, or add different conjugates of PUFAs, in orderto prevent, control or overcome such mechanisms to achieve the desiredlevels of specific PUFAs in an individual.

In alternate embodiments, PUFAs, or derivatives thereof, made by themethodology disclosed herein can be utilized in the synthesis ofaquaculture feeds (i.e., dry feeds, semi-moist and wet feeds) sincethese formulations generally require at least 1-2% of the nutrientcomposition to be ω-3 and/or ω-6 PUFAs.

DEFINITIONS

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

“Diacylglycerol acyltransferase” is abbreviated DAG AT or DGAT.

“Phospholipid:diacylglycerol acyltransferase” is abbreviated PDAT.

“Glycerol-3-phosphate acyltransferase” is abbreviated GPAT.

“Lysophosphatidic acid acyltransferase” is abbreviated LPAAT.

“Acyl-CoA:1-acyl lysophosphatidylcholine acyltransferase” is abbreviated“LPCAT”.

“Acyl-CoA:sterol-acyltransferase” is abbreviated ARE2.

“Diacylglycerol” is abbreviated DAG.

“Triacylglycerols” are abbreviated TAGs.

“Co-enzyme A” is abbreviated CoA.

“Phosphatidyl-choline” is abbreviated PC.

The term “Fusarium moniliforme” is synonymous with “Fusariumverticillioides”.

The term “food product” refers to any food generally suitable for humanconsumption. Typical food products include but are not limited to meatproducts, cereal products, baked foods, snack foods, dairy products andthe like.

The term “functional food” refers to those foods that encompasspotentially healthful products including any modified food or ingredientthat may provide a health benefit beyond the traditional nutrients itcontains. Functional foods can include foods like cereals, breads andbeverages which are fortified with vitamins, herbs and nutraceuticals.Functional foods contain a substance that provides health benefitsbeyond its nutritional value, wherein the substance either is naturallypresent in the food or is deliberately added.

As used herein the term “medical food” refers to a food which isformulated to be consumed or administered enterally under thesupervision of a physician and which is intended for the specificdietary management of a disease or condition for which distinctivenutritional requirements, based on recognized scientific principles, areestablished by medical evaluation [see section 5(b) of the Orphan DrugAct (21 U.S.C. 360ee(b)(3))]. A food is a “medical food” only if: (i) Itis a specially formulated and processed product (as opposed to anaturally occurring foodstuff used in its natural state) for the partialor exclusive feeding of a patient by means of oral intake or enteralfeeding by tube; (ii) It is intended for the dietary management of apatient who, because of therapeutic or chronic medical needs, haslimited or impaired capacity to ingest, digest, absorb, or metabolizeordinary foodstuffs or certain nutrients, or who has other specialmedically determined nutrient requirements, the dietary management ofwhich cannot be achieved by the modification of the normal diet alone;(iii) It provides nutritional support specifically modified for themanagement of the unique nutrient needs that result from the specificdisease or condition, as determined by medical evaluation; (iv) It isintended to be used under medical supervision; and (v) It is intendedonly for a patient receiving active and ongoing medical supervisionwherein the patient requires medical care on a recurring basis for,among other things, instructions on the use of the medical food. Thus,unlike dietary supplements or conventional foods, a medical food that isintended for the specific dietary management of a disease or conditionfor which distinctive nutritional requirements have been established,may bear scientifically valid claims relating to providing distinctivenutritional support for a specific disease or condition. Medical foodsare distinguished from the broader category of foods for special dietaryuse (e.g., hypoallergenic foods) and from foods that make health claims(e.g., dietary supplements) by the requirement that medical foods beused under medical supervision.

The term “medical nutritional” is a medical food as defined hereintypically refers to a fortified beverage that is specifically designedfor special dietary needs. The medical nutritional generally comprises adietary composition focused at a specific medical or dietary condition.Examples of commercial medical nuturitionals include, but are notlimited to Ensure® and Boost®.

The term “pharmaceutical” as used herein means a compound or substancewhich if sold in the United States would be controlled by Section 505 or505 of the Federal Food, Drug and Cosmetic Act.

The term “infant formula” means a food which is designed exclusively forconsumption by the human infant by reason of its simulation of humanbreast milk. Typical commercial examples of infant formula include burare not limited to Similac®, and Isomil®.

The term “dietary supplement” refers to a product that: (i) is intendedto supplement the diet and thus is not represented for use as aconventional food or as a sole item of a meal or the diet; (ii) containsone or more dietary ingredients (including, e.g., vitamins, minerals,herbs or other botanicals, amino acids, enzymes and glandulars) or theirconstituents; (iii) is intended to be taken by mouth as a pill, capsule,tablet, or liquid; and (iv) is labeled as being a dietary supplement.

A “food analog” is a food-like product manufactured to resemble its foodcounterpart, whether meat, cheese, milk or the like, and is intended tohave the appearance, taste, and texture of its counterpart. Thus, theterm “food” as used herein also encompasses food analogs.

The terms “aquaculture feed” and “aquafeed” refer to manufactured orartificial diets (formulated feeds) to supplement or to replace naturalfeeds in the aquaculture industry. Thus, an aquafeed refers toartificially compounded feeds that are useful for farmed finfish andcrustaceans (i.e., both lower-value staple food fish species [e.g.,freshwater finfish such as carp, tilapia and catfish] and higher-valuecash crop species for luxury or niche markets [e.g., mainly marine anddiadromous species such as shrimp, salmon, trout, yellowtail, seabass,seabream and grouper]). These formulate feeds are composed of severalingredients in various proportions complementing each other to form anutritionally complete diet for the aquacultured species.

The term “animal feed” refers to feeds intended exclusively forconsumption by animals, including domestic animals (pets, farm animalsetc.) or for animals raised for the production of food e.g. fishfarming.

The term “feed nutrient” means nutrients such as proteins, lipids,carbohydrates, vitamins, minerals, and nucleic acids that may be derivedfrom the yeast biomass comprising the recombinant production hosts ofthe invention.

As used herein the term “biomass” refers specifically to spent or usedyeast cellular material from the fermentation of a recombinantproduction host production EPA in commercially significant amounts.

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain lengths, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon (C) atoms in the particular fatty acid and Y is thenumber of double bonds. Additional details concerning thedifferentiation between “saturated fatty acids” versus “unsaturatedfatty acids”, “monounsaturated fatty acids” versus “polyunsaturatedfatty acids” (or “PUFAs”), and “omega-6 fatty acids” (ω-6 or n-6) versus“omega-3 fatty acids” (ω-3 or n-3) are provided in WO2004/101757.

Nomenclature used to describe PUFAs in the present disclosure is shownbelow in Table 3. In the column titled “Shorthand Notation”, theomega-reference system is used to indicate the number of carbons, thenumber of double bonds and the position of the double bond closest tothe omega carbon, counting from the omega carbon (which is numbered 1for this purpose). The remainder of the Table summarizes the commonnames of ω-3 and ω-6 fatty acids and their precursors, the abbreviationsthat will be used throughout the specification and each compounds'chemical name.

TABLE 3 Nomenclature of Polyunsaturated Fatty Acids And PrecursorsShorthand Common Name Abbreviation Chemical Name Notation Myristic —tetradecanoic 14:0 Palmitic Palmitate hexadecanoic 16:0 Palmitoleic —9-hexadecenoic 16:1 Stearic — octadecanoic 18:0 Oleic — cis-9- 18:1octadecenoic Linoleic LA cis-9,12- 18:2 ω-6 octadecadienoic γ-LinoleicGLA cis-6,9,12- 18:3 ω-6 octadecatrienoic Eicosadienoic EDA cis-11,14-20:2 ω-6 eicosadienoic Dihomo-γ- DGLA cis-8,11,14- 20:3 ω-6 Linoleiceicosatrienoic Arachidonic ARA cis-5,8,11,14- 20:4 ω-6 eicosatetraenoicα-Linolenic ALA cis-9,12,15- 18:3 ω-3 octadecatrienoic Stearidonic STAcis-6,9,12,15- 18:4 ω-3 octadecatetraenoic Eicosatrienoic ETrAcis-11,14,17- 20:3 ω-3 eicosatrienoic Eicosa- ETA cis-8,11,14,17- 20:4ω-3 tetraenoic eicosatetraenoic Eicosa- EPA cis-5,8,11,14,17- 20:5 ω-3pentaenoic eicosapentaenoic Docosa- DPA cis-7,10,13,16,19- 22:5 ω-3pentaenoic docosapentaenoic Docosa- DHA cis-4,7,10,13,16,19- 22:6 ω-3hexaenoic docosahexaenoic

The term “high-level ARA production” refers to production of at leastabout 5% ARA in the total lipids of the microbial host, preferably atleast about 10% ARA in the total lipids, more preferably at least about15% ARA in the total lipids, more preferably at least about 20% ARA inthe total lipids and most preferably at least about 25-30% ARA in thetotal lipids. The structural form of the ARA is not limiting; thus, forexample, the ARA may exist in the total lipids as free fatty acids or inesterified forms such as acylglycerols, phospholipids, sulfolipids orglycolipids.

The term “devoid of any GLA” refers to lack of any detectable GLA in thetotal lipids of the microbial host, when measured by GC analysis usingequipment having a detectable level down to about 0.1%.

The term “essential fatty acid” refers to a particular PUFA that anorganism must ingest in order to survive, being unable to synthesize theparticular essential fatty acid de novo. For example, mammals can notsynthesize the essential fatty acids LA (18:2, ω-6) and ALA (18:3, ω-3).Other essential fatty acids include GLA (ω-6), DGLA (ω-6), ARA (ω-6),EPA (ω-3) and DHA (ω-3).

“Microbial oils” or “single cell oils” are those oils naturally producedby microorganisms (e.g., algae, oleaginous yeasts and filamentous fungi)during their lifespan. The term “oil” refers to a lipid substance thatis liquid at 25° C. and usually polyunsaturated. In contrast, the term“fat” refers to a lipid substance that is solid at 25° C. and usuallysaturated.

“Lipid bodies” refer to lipid droplets that usually are bounded byspecific proteins and a monolayer of phospholipid. These organelles aresites where most organisms transport/store neutral lipids. Lipid bodiesare thought to arise from microdomains of the endoplasmic reticulum thatcontain TAG-biosynthesis enzymes; and, their synthesis and size appearto be controlled by specific protein components.

“Neutral lipids” refer to those lipids commonly found in cells in lipidbodies as storage fats and oils and are so called because at cellularpH, the lipids bear no charged groups. Generally, they are completelynon-polar with no affinity for water. Neutral lipids generally refer tomono-, di-, and/or triesters of glycerol with fatty acids, also calledmonoacylglycerol, diacylglycerol or TAG, respectively (or collectively,acylglycerols). A hydrolysis reaction must occur to release free fattyacids from acylglycerols.

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

The term “acyltransferase” refers to an enzyme responsible fortransferring a group other than an amino-acyl group (EC 2.3.1.-).

The term “DAG AT” refers to a diacylglycerol acyltransferase (also knownas an acyl-CoA-diacylglycerol acyltransferase or a diacylglycerolO-acyltransferase) (EC 2.3.1.20). This enzyme is responsible for theconversion of acyl-CoA and 1,2-diacylglycerol to TAG and CoA (therebyinvolved in the terminal step of TAG biosynthesis). Two families of DAGAT enzymes exist: DGAT1 and DGAT2. The former family shares homologywith the acyl-CoA:cholesterol acyltransferase (ACAT) gene family, whilethe latter family is unrelated (Lardizabal et al., J. Biol. Chem.276(42):38862-38869 (2001)).

The term “PDAT” refers to a phospholipid:diacylglycerol acyltransferaseenzyme (EC 2.3.1.158). This enzyme is responsible for the transfer of anacyl group from the sn-2 position of a phospholipid to the sn-3 positionof 1,2-diacylglycerol, thus resulting in lysophospholipid and TAG(thereby involved in the terminal step of TAG biosynthesis). This enzymediffers from DGAT (EC 2.3.1.20) by synthesizing TAG via anacyl-CoA-independent mechanism.

The term “ARE2” refers to an acyl-CoA:sterol-acyltransferase enzyme (EC2.3.1.26; also known as a sterol-ester synthase 2 enzyme), catalyzingthe following reaction: acyl-CoA+sterol=CoA+sterol ester.

The term “GPAT” refers to a glycerol-3-phosphate O-acyltransferaseenzyme (E.C. 2.3.1.15) encoded by the gpat gene and which convertsacyl-CoA and sn-glycerol 3-phosphate to CoA and 1-acyl-sn-glycerol3-phosphate (the first step of phospholipid biosynthesis).

The term “LPAAT” refers to a lysophosphatidic acid-acyltransferaseenzyme (EC 2.3.1.51). This enzyme is responsible for the transfer of anacyl-CoA group onto 1-acyl-sn-glycerol 3-phosphate (i.e.,lysophosphatidic acid) to produce CoA and 1,2-diacyl-sn-glycerol3-phosphate (phosphatidic acid). The literature also refers to LPAAT asacyl-CoA:1-acyl-sn-glycerol-3-phosphate 2-O-acyltransferase,1-acyl-sn-glycerol-3-phosphate acyltransferase and/or1-acylglycerolphosphate acyltransferase (abbreviated as AGAT).

The term “LPCAT” refers to an acyl-CoA:1-acyl lysophosphatidyl-cholineacyltransferase. This enzyme is responsible for the exchange of acylgroups between CoA and phosphatidyl choline (PC). Herein it also refersto enzymes involved the acyl exchange between CoA and otherphospholipids, including lysophosphatidic acid (LPA).

“Percent (%) PUFAs in the total lipid and oil fractions” refers to thepercent of PUFAs relative to the total fatty acids in those fractions.The term “total lipid fraction” or “lipid fraction” both refer to thesum of all lipids (i.e., neutral and polar) within an oleaginousorganism, thus including those lipids that are located in thephosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fractionand triacylglycerol (TAG or oil) fraction. However, the terms “lipid”and “oil” will be used interchangeably throughout the specification.

The term “phosphatidylcholine” or “PC” refers to a phospholipid that isa major constituent of cell membranes. The chemical structure of PC cangenerally be described as comprising the following: a choline molecule,a phosphate group and glycerol, wherein fatty acyl chains are attachedas R groups on the sn-1 and sn-2 positions of the glycerol molecule.

The term “PUFA biosynthetic pathway enzyme” refers to any of thefollowing enzymes (and genes which encode said enzymes) associated withthe biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase,a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, aΔ9 desaturase, a Δ8 desaturase, a Δ9 elongase, a C_(14/16) elongase, aC_(16/18) elongase, a C_(18/20) elongase and/or a C_(20/22) elongase.

The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set ofgenes which, when expressed under the appropriate conditions encodeenzymes that catalyze the production of either or both ω-3 and ω-6 fattyacids. Typically the genes involved in the ω-3/ω-6 fatty acidbiosynthetic pathway encode some or all of the following enzymes: Δ12desaturase, Δ6 desaturase, C_(18/20) elongase, C_(20/22) elongase, Δ9elongase, Δ5 desaturase, Δ17 desaturase, Δ15 desaturase, Δ9 desaturase,Δ8 desaturase, and Δ4 desaturase. A representative pathway isillustrated in FIG. 1, providing for the conversion of oleic acidthrough various intermediates to DHA, which demonstrates how both ω-3and ω-6 fatty acids may be produced from a common source. The pathway isnaturally divided into two portions where one portion will generate ω-3fatty acids and the other portion, only ω-6 fatty acids. That portionthat only generates ω-3 fatty acids will be referred to herein as theω-3 fatty acid biosynthetic pathway, whereas that portion that generatesonly ω-6 fatty acids will be referred to herein as the ω-6 fatty acidbiosynthetic pathway.

The term “functional” as used herein in context with the ω-3/ω-6 fattyacid biosynthetic pathway means that some (or all of) the genes in thepathway express active enzymes, resulting in in vivo catalysis orsubstrate conversion. It should be understood that “ω-3/ω)-6 fatty acidbiosynthetic pathway” or “functional ω-3/ω)-6 fatty acid biosyntheticpathway” does not imply that all the genes listed in the above paragraphare required, as a number of fatty acid products will only require theexpression of a subset of the genes of this pathway.

The term “Δ6 desaturase/Δ6 elongase pathway” will refer to an ARA fattyacid biosynthetic pathway that minimally includes the following genes:Δ6 desaturase, C_(18/20) elongase and Δ5 desaturase. In a relatedmanner, the term “Δ9 elongase/Δ8 desaturase pathway” will refer to anARA fatty acid biosynthetic pathway that minimally includes thefollowing genes: Δ9 elongase, Δ8 desaturase and Δ5 desaturase.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a fattyacid or precursor of interest. Despite use of the omega-reference systemthroughout the specification to refer to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Ofparticular interest herein are: 1.) Δ8 desaturases that desaturate afatty acid between the 8^(th) and 9^(th) carbon atom numbered from thecarboxyl-terminal end of the molecule and which, for example, catalyzethe conversion of EDA to DGLA and/or ETrA to ETA; 2.) Δ6 desaturasesthat catalyze the conversion of LA to GLA and/or ALA to STA; 3.) Δ5desaturases that catalyze the conversion of DGLA to ARA and/or ETA toEPA; 4.) Δ4 desaturases that catalyze the conversion of DPA to DHA; 5.)Δ12 desaturases that catalyze the conversion of oleic acid to LA; 6.)Δ15 desaturases that catalyze the conversion of LA to ALA and/or GLA toSTA; 7.) Δ17 desaturases that catalyze the conversion of ARA to EPAand/or DGLA to ETA; and 8.) Δ9 desaturases that catalyze the conversionof palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid(18:1).

The term “bifunctional” as it refers to Δ15 desaturases of the inventionmeans that the polypeptide has the ability to use both oleic acid andlinoleic acid as an enzymatic substrate. By “enzymatic substrate” it ismeant that the polypeptide binds the substrate at an active site andacts upon it in a reactive manner.

The term “elongase system” refers to a suite of four enzymes that areresponsible for elongation of a fatty acid carbon chain to produce afatty acid that is 2 carbons longer than the fatty acid substrate thatthe elongase system acts upon. More specifically, the process ofelongation occurs in association with fatty acid synthase, whereby CoAis the acyl carrier (Lassner et al., The Plant Cell 8:281-292 (1996)).In the first step, which has been found to be both substrate-specificand also rate-limiting, malonyl-CoA is condensed with a long-chainacyl-CoA to yield CO₂ and a β-ketoacyl-CoA (where the acyl moiety hasbeen elongated by two carbon atoms). Subsequent reactions includereduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a secondreduction to yield the elongated acyl-CoA. Examples of reactionscatalyzed by elongase systems are the conversion of GLA to DGLA, STA toETA and EPA to DPA.

For the purposes herein, an enzyme catalyzing the first condensationreaction (i.e., conversion of malonyl-CoA to 3-ketoacyl-CoA) will bereferred to generically as an “elongase”. In general, the substrateselectivity of elongases is somewhat broad but segregated by both chainlength and the degree and type of unsaturation. Accordingly, elongasescan have different specificities. For example, a C_(14/16) elongase willutilize a C₁₄ substrate (e.g., myristic acid), a C_(16/18) elongase willutilize a C₁₆ substrate (e.g., palmitate), a C_(18/20) elongase willutilize a C₁₈ substrate (e.g., GLA, STA) and a C_(20/22) elongase willutilize a C₂₀ substrate (e.g., EPA). In like manner, a Δ9 elongase isable to catalyze the conversion of LA and ALA to EDA and ETrA,respectively. It is important to note that some elongases have broadspecificity and thus a single enzyme may be capable of catalyzingseveral elongase reactions (e.g., thereby acting as both a C_(16/18)elongase and a C_(18/20) elongase). In preferred embodiments, it is mostdesirable to empirically determine the specificity of a fatty acidelongase by transforming a suitable host with the gene for the fattyacid elongase and determining its effect on the fatty acid profile ofthe host.

The term “high affinity elongase” or “EL1S” or “ELO1” refers to aC_(18/20) elongase whose substrate specificity is preferably for GLA(with DGLA as a product of the elongase reaction [i.e., a Δ6 elongase]).One such elongase is described in WO 00/12720 and is provided herein asSEQ ID NOs:17 and 18. However, the Applicants have shown that thisenzyme also has some activity on 18:2 (LA) and 18:3 (ALA); thus, SEQ IDNO:18 shows Δ9 elongase activity (in addition to its Δ6 elongaseactivity). It is therefore concluded that the C_(18/20) elongaseprovided herein as SEQ ID NO:18 can function both within the Δ6desaturase/Δ6 elongase pathway as described in the invention herein andwithin the Δ9 elongase/Δ8 desaturase pathway, as a substitute for e.g.,the Isochrysis galbana Δ9 elongase (SEQ ID NO:40).

The term “EL2S” or “ELO2” refers to a C_(18/20) elongase whose substratespecificity is preferably for GLA (with DGLA as a product of theelongase reaction) and/or STA (with STA as a product of the elongasereaction). One such elongase is described in U.S. Pat. No. 6,677,145 andis provided herein as SEQ ID NOs:20 and 21.

The term “ELO3” refers to a Mortierella alpine C_(16/18) fatty acidelongase enzyme (provided herein as SEQ ID NO:54), encoded by the elo3gene (SEQ ID NO:53). The term “YE2” refers to a Yarrowia lipolyticaC_(16/18) fatty acid elongase enzyme (provided herein as SEQ ID NO:62),encoded by the gene provided herein as SEQ ID NO:61. Based on datareported herein, both ELO3 and YE2 preferentially catalyze theconversion of palmitate (16:0) to stearic acid (18:0).

The term “YE1” refers to a Yarrowia lipolytica C_(14/16) fatty acidelongase enzyme (provided herein as SEQ ID NO:65), encoded by the geneprovided herein as SEQ ID NO:64. Based on data reported herein, YE2preferentially catalyzes the conversion of myristic acid (14:0) topalmitate (16:0).

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., a desaturaseor elongase) can convert substrate to product. The conversion efficiencyis measured according to the following formula:

([product]/[substrate+product])*100,

where ‘product’ includes the immediate product and all products in thepathway derived from it.

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2^(nd) Ed., Plenum, 1980). Generally, the cellular oilcontent of these microorganisms follows a sigmoid curve, wherein theconcentration of lipid increases until it reaches a maximum at the latelogarithmic or early stationary growth phase and then graduallydecreases during the late stationary and death phases (Yongmanitchai andWard, Appl. Environ. Microbiol. 57:419-25 (1991)).

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that can make oil. Generally, the cellular oil or triacylglycerolcontent of oleaginous microorganisms follows a sigmoid curve, whereinthe concentration of lipid increases until it reaches a maximum at thelate logarithmic or early stationary growth phase and then graduallydecreases during the late stationary and death phases (Yongmanitchai andWard, Appl. Environ. Microbiol. 57:419-25 (1991)). It is not uncommonfor oleaginous microorganisms to accumulate in excess of about 25% oftheir dry cell weight as oil. Examples of oleaginous yeast include, butare no means limited to, the following genera: Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

The term “fermentable carbon source” means a carbon source that amicroorganism will metabolize to derive energy. Typical carbon sourcesof the invention include, but are not limited to: monosaccharides,oligosaccharides, polysaccharides, alkanes, fatty acids, esters of fattyacids, monoglycerides, diglycerides, triglycerides, carbon dioxide,methanol, formaldehyde, formate and carbon-containing amines.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)).In general, a sequence of ten or more contiguous amino acids or thirtyor more nucleotides is necessary in order to identify putatively apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Chemically synthesized”, as related to a sequence of DNA, means thatthe component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures;or, automated chemical synthesis can be performed using one of a numberof commercially available machines. “Synthetic genes” can be assembledfrom oligonucleotide building blocks that are chemically synthesizedusing procedures known to those skilled in the art. These buildingblocks are ligated and annealed to form gene segments that are thenenzymatically assembled to construct the entire gene. Accordingly, thegenes can be tailored for optimal gene expression based on optimizationof nucleotide sequence to reflect the codon bias of the host cell. Theskilled artisan appreciates the likelihood of successful gene expressionif codon usage is biased towards those codons favored by the host.Determination of preferred codons can be based on a survey of genesderived from the host cell, where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and that may refer to the coding region alone or may includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene that is introduced intothe host organism by gene transfer. Foreign genes can comprise nativegenes inserted into a non-native organism, native genes introduced intoa new location within the native host, or chimeric genes. A “transgene”is a gene that has been introduced into the genome by a transformationprocedure. A “codon-optimized gene” is a gene having its frequency ofcodon usage designed to mimic the frequency of preferred codon usage ofthe host cell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “GPAT promoter” or “GPAT promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a glycerol-3-phosphate O-acyltransferase enzyme(E.C. 2.3.1.15) encoded by the gpat gene and that is necessary forexpression. Examples of suitable Yarrowia lipolytica GPAT promoterregions are described in U.S. patent application Ser. No. 11/225,354.

The term “GPD promoter” or “GPD promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a glyceraldehyde-3-phosphate dehydrogenase enzyme(E.C. 1.2.1.12) encoded by the gpd gene and that is necessary forexpression. Examples of suitable Yarrowia lipolytica GPD promoterregions are described in WO 2005/003310.

The term “GPM promoter” or “GPM promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a phosphoglycerate mutase enzyme (EC 5.4.2.1)encoded by the gpm gene and that is necessary for expression. Examplesof suitable Yarrowia lipolytica GPM promoter regions are described in WO2005/003310.

The term “FBA promoter” or “FBA promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a fructose-bisphosphate aldolase enzyme (E.C.4.1.2.13) encoded by the fba1 gene and that is necessary for expression.Examples of suitable Yarrowia lipolytica FBA promoter regions aredescribed in WO 2005/049805.

The term “FBAIN promoter” or “FBAIN promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of the fba1 gene and that is necessary for expression,plus a portion of 5′ coding region that has an intron of the fba1 gene.Examples of suitable Yarrowia lipolytica FBAIN promoter regions aredescribed in WO 2005/049805.

The term “GPDIN promoter” or “GPDIN promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of the gpd gene and that is necessary for expression,plus a portion of 5′ coding region that has an intron of the gpd gene.Examples of suitable Yarrowia lipolytica GPDIN promoter regions aredescribed in U.S. patent application Ser. No. 11/183,664.

The term “YAT1 promoter” or “YAT1 promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of an ammonium transporter enzyme (TC 2.A.49; GenBankAccession No. XM_(—)504457) encoded by the yat1 gene and that isnecessary for expression. Examples of suitable Yarrowia lipolytica YAT1promoter regions are described in U.S. patent application Ser. No.11/185,301.

The term “EXP1 promoter” or “EXP1 promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a protein encoded by the Yarrowia lipolytica“YALI0C12034g” gene (GenBank Accession No. XM_(—)501745) and that isnecessary for expression. Based on significant homology of“YALI0C12034g” to the sp|Q12207 S. cerevisiae non-classical exportprotein 2 (whose function is involved in a novel pathway of export ofproteins that lack a cleavable signal sequence), this gene is hereindesignated as the exp1 gene, encoding a protein designated as EXP1. Anexample of a suitable Yarrowia lipolytica EXP1 promoter region isdescribed as SEQ ID NO:364, but this is not intended to be limiting innature. One skilled in the art will recognize that since the exactboundaries of the EXP1 promoter sequence have not been completelydefined, DNA fragments of increased or diminished length may haveidentical promoter activity.

The term “promoter activity” will refer to an assessment of thetranscriptional efficiency of a promoter. This may, for instance, bedetermined directly by measurement of the amount of mRNA transcriptionfrom the promoter (e.g., by Northern blotting or primer extensionmethods) or indirectly by measuring the amount of gene product expressedfrom the promoter.

“Introns” are sequences of non-coding DNA found in gene sequences(either in the coding region, 5′ non-coding region, or 3′ non-codingregion) in most eukaryotes. Their full function is not known; however,some enhancers are located in introns (Giacopelli F. et al., Gene Expr.11: 95-104 (2003)). These intron sequences are transcribed, but removedfrom within the pre-mRNA transcript before the mRNA is translated into aprotein. This process of intron removal occurs by self-splicing of thesequences (exons) on either side of the intron.

The term “enhancer” refers to a cis-regulatory sequence that can elevatelevels of transcription from an adjacent eukaryotic promoter, therebyincreasing transcription of the gene. Enhancers can act on promotersover many tens of kilobases of DNA and can be 5′ or 3′ to the promoterthey regulate. Enhancers can also be located within introns.

The terms “3′ non-coding sequences” and “transcription terminator” referto DNA sequences located downstream of a coding sequence. This includespolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The 3′ region can influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” or “mRNA” refersto the RNA that is without introns and that can be translated intoprotein by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to, and derived from, mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO99/28508). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that is not translated and yethas an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragments of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be (but are not limited to)intracellular localization signals.

The term “recombinase” refers to an enzyme(s) that carries outsite-specific recombination to alter the DNA structure and includestransposases, lambda integration/excision enzymes, as well assite-specific recombinases.

“Recombinase site” or “site-specific recombinase sequence” means a DNAsequence that a recombinase will recognize and bind to. It will beappreciated that this may be a wild type or mutant recombinase site, aslong as functionality is maintained and the recombinase enzyme may stillrecognize the site, bind to the DNA sequence, and catalyze therecombination between two adjacent recombinase sites.

“Transformation” refers to the transfer of a nucleic acid molecule intoa host organism, resulting in genetically stable inheritance. Thenucleic acid molecule may be a plasmid that replicates autonomously, forexample; or, it may integrate into the genome of the host organism. Hostorganisms containing the transformed nucleic acid fragments are referredto as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Expression cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that allow for enhanced expression of that gene in a foreign host.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules (during cross over). The fragmentsthat are exchanged are flanked by sites of identical nucleotidesequences between the two DNA molecules (i.e., “regions of homology”).The term “regions of homology” refer to stretches of nucleotide sequenceon nucleic acid fragments that participate in homologous recombinationthat have homology to each other. Effective homologous recombinationwill generally take place where these regions of homology are at leastabout 10 bp in length where at least about 50 bp in length is preferred.Typically fragments that are intended for recombination contain at leasttwo regions of homology where targeted gene disruption or replacement isdesired.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialin the structure, the stability, or the activity of a protein. Becausethey are identified by their high degree of conservation in alignedsequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family. Amotif that is indicative of a fungal protein having Δ15 desaturaseactivity is provided as SEQ ID NO:374, while a motif that is indicativeof a fungal protein having Δ12 desaturase activity is provided as SEQ IDNO:375.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist,L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

A Preferred Microbial Host for ARA Production: Yarrowia lipolytica

Prior to work by the Applicants (see, Picataggio et al., WO2004/101757),oleaginous yeast have not been examined previously as a class ofmicroorganisms suitable for use as a production platform for PUFAs.Genera typically identified as oleaginous yeast include, but are notlimited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces. More specifically,illustrative oil-synthesizing yeast include: Rhodosporidium toruloides,Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C.tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorulaglutinus, R. graminis and Yarrowia lipolytica (formerly classified asCandida lipolytica).

Oleaginous yeast were considered to have several qualities that wouldfacilitate their use as a host organism for economical, commercialproduction of ARA. First, the organisms are defined as those that arenaturally capable of oil synthesis and accumulation, wherein the oil cancomprise greater than about 25% of the cellular dry weight, morepreferably greater than about 30% of the cellular dry weight and mostpreferably greater than about 40% of the cellular dry weight. Secondly,the technology for growing oleaginous yeast with high oil content iswell developed (for example, see EP 0 005 277B1; Ratledge, C., Prog.Ind. Microbiol. 16:119-206 (1982)). And, these organisms have beencommercially used for a variety of purposes in the past. For example,various strains of Yarrowia lipolytica have historically been used forthe manufacture and production of: isocitrate lyase (DD259637); lipases(SU1454852, WO2001083773, DD279267); polyhydroxyalkanoates(WO2001088144); citric acid (RU2096461, RU2090611, DD285372, DD285370,DD275480, DD227448, PL160027); erythritol (EP770683); 2-oxoglutaric acid(DD267999); γ-decalactone (U.S. Pat. No. 6,451,565, FR2734843);γ-dodecalactone (EP578388); and pyruvic acid (JP09252790).

Of those organisms classified as oleaginous yeast, Yarrowia lipolyticawas selected as the preferred microbial host for the purposes herein.This selection was based on the knowledge that oleaginous strains wereavailable that were capable of incorporating ω-6 and ω-3 fatty acidsinto the TAG fraction, the organism was amenable to geneticmanipulation, and previous use of the species as a Generally RecognizedAs Safe (“GRAS”, according to the U.S. Food and Drug Administration)source of food-grade citric acid. In a further embodiment, mostpreferred are the Y. lipolytica strains designated as ATCC #20362, ATCC#8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., andAggelis G., Bioresour. Technol. 82(1):43-9 (2002)), due to preliminarystudies targeted toward identification of wildtype strains having highlipid content (measured as a percent dry weight) and high volumetricproductivity (measured as g/L h⁻¹).

As described in WO 2004/101757, Yarrowia lipolytica was previouslygenetically engineered to produce 1.3% ARA and 1.9% EPA, respectively,by introduction and expression of genes encoding the ω-3/ω-6biosynthetic pathway. More specifically, two different DNA expressionconstructs (comprising either a Δ6 desaturase, Δ5 desaturase andhigh-affinity PUFA C_(18/20) elongase for ARA synthesis or a Δ6desaturase, Δ5 desaturase, high-affinity PUFA C_(18/20) elongase andcodon-optimized Δ17 desaturase for EPA synthesis) were separatelytransformed and integrated into the Y. lipolytica chromosomal URA3 geneencoding the enzyme orotidine-5′-phosphate decarboxylase (EC 4.1.1.23).GC analysis of the host cells fed with appropriate substrates detectedproduction of ARA and EPA. Although suitable to demonstrateproof-of-concept for the ability of oleaginous hosts to be geneticallyengineered for production of ω-6 and ω-3 fatty acids, this work failedto perform the complex metabolic engineering required to enablesynthesis of greater than 5% ARA in the total oil fraction, or morepreferably greater than 10% ARA in the total oil fraction, or even morepreferably greater than 15-20% ARA in the total oil fraction, or mostpreferably greater than 25-30% ARA in the total oil fraction.

In co-pending U.S. Patent Application No. 60/624,812, complex metabolicengineering within Yarrowia lipolytica was performed to: (1) identifypreferred desaturases and elongases that allow for the synthesis andhigh accumulation of EPA; (2) manipulate the activity ofacyltransferases that allow for the transfer of omega fatty acids intostorage lipid pools; (3) over-express desaturases, elongases andacyltransferases by use of strong promoters, expression in multicopy,and/or codon-optimization; (4) down-regulate the expression of specificgenes within the PUFA biosynthetic pathway that diminish overallaccumulation of EPA; and, (5) manipulate pathways and global regulatorsthat affect EPA production. This resulted in the production of up to 28%EPA in one particular recombinant strain of Yarrowia lipolytica.

In the present Application, analogous complex metabolic engineering isperformed to result in the production of 10-14% ARA in the total oilfraction in recombinant strains of Yarrowia lipolytica. Morespecifically, strains Y2034 and Y2047 were genetically engineered toutilize the Δ6 desaturase/Δ6 elongase pathway and produced oilcomprising 10% ARA and 11% ARA, respectively; strain Y2214 wasgenetically engineered to utilize the Δ9 elongase/Δ8 desaturase pathwayand produced oil comprising 14% ARA that was devoid of GLA. Aspects ofthe metabolic engineering utilized will be discussed below, as willadditional engineering and fermentation methods that could be performedto further enhance ARA productivity in this oleaginous yeast.

An Overview Microbial Biosynthesis of Fatty Acids and Triacylglycerols

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium. This process, leading to the de novo synthesis of freepalmitate (16:0) in oleaginous microorganisms, is described in detail inWO 2004/101757. Palmitate is the precursor of longer-chain saturated andunsaturated fatty acid derivates, which are formed through the action ofelongases and desaturases. For example, palmitate is converted to itsunsaturated derivative [palmitoleic acid (16:1)] by the action of a Δ9desaturase; similarly, palmitate is elongated by a C_(16/18) fatty acidelongase to form stearic acid (18:0), which can be converted to itsunsaturated derivative by a Δ9 desaturase to thereby yield oleic (18:1)acid.

TAGs (the primary storage unit for fatty acids) are formed by a seriesof reactions that involve: 1.) the esterification of one molecule ofacyl-CoA to glycerol-3-phosphate via an acyltransferase to producelysophosphatidic acid; 2.) the esterification of a second molecule ofacyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate(commonly identified as phosphatidic acid); 3.) removal of a phosphateby phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and4.) the addition of a third fatty acid by the action of anacyltransferase to form TAG (FIG. 2).

A wide spectrum of fatty acids can be incorporated into TAGs, includingsaturated and unsaturated fatty acids and short-chain and long-chainfatty acids. Some non-limiting examples of fatty acids that can beincorporated into TAGs by acyltransferases include: capric (10:0),lauric (12:0), myristic (14:0), palmitic (16:0), palmitoleic (16:1),stearic (18:0), oleic (18:1), vaccenic (18:1), LA, eleostearic (18:3),ALA, GLA, arachidic (20:0), EDA, ETrA, DGLA, ETA, ARA, EPA, behenic(22:0), DPA, DHA, lignoceric (24:0), nervonic (24:1), cerotic (26:0),and montanic (28:0) fatty acids. In preferred embodiments of the presentinvention, incorporation of ARA into TAG is most desirable.

Biosynthesis of ARA, an ω-6 Fatty Acid

The metabolic process wherein oleic acid is converted to ARA involveselongation of the carbon chain through the addition of carbon atoms anddesaturation of the molecule through the addition of double bonds. Thisrequires a series of special desaturation and elongation enzymes presentin the endoplasmic reticulim membrane. However, as seen in FIG. 1 and asdescribed below, two alternate pathways exist for ARA production.

Specifically, both pathways require the initial conversion of oleic acidto LA (18:2), the first of the ω-6 fatty acids, by the action of a Δ12desaturase. Then, using the “Δ6 desaturase/Δ6 elongase pathway” for ARAbiosynthesis, PUFAs are formed as follows: (1) LA is converted to GLA bythe activity of a Δ6 desaturase; (2) GLA is converted to DGLA by theaction of a C_(18/20) elongase; and (3) DGLA is converted to ARA by theaction of a Δ5 desaturase.

Alternatively, via the “Δ9 elongase/Δ8 desaturase pathway”, LA isconverted to EDA by the action of a Δ9 elongase; then, a Δ8 desaturaseconverts EDA to DGLA. Subsequent desaturation of DGLA by the action of aΔ5 desaturase yields ARA, as described above.

For the sake of clarity, each of these pathways will be summarized inthe Table below, as well as their distinguishing characteristics:

TABLE 4 Alternate Biosynthetic Pathways For ARA Biosynthesis MinimumRequired Name Genes For ARA* Pathway Δ6 desaturase/Δ6 elongase Δ6D,C_(18/20) — pathway ELO, Δ5D Δ9 elongase/Δ8 desaturase Δ9 ELO, producesoil that pathway Δ8D, Δ5D is devoid of GLA Combination Δ6 desaturase/Δ6Δ6D, C_(18/20) — elongase and Δ9 elongase/Δ8 ELO, Δ9 ELO, desaturasepathway Δ8D, Δ5D *Abbreviations: “D” = desaturase; “ELO” = elongase.

If desirable, several other PUFAs can be produced using ARA assubstrate. For example, ARA can be further desaturated to EPA by a Δ17desaturase and subsequently converted to DHA by the action of aC_(20/22) elongase and a Δ4 desaturase.

Selection of Microbial Genes for ARA Synthesis

The particular functionalities required to be introduced into Yarrowialipolytica for production of ARA will depend on the host cell (and itsnative PUFA profile and/or desaturase/elongase profile), theavailability of substrate, and the desired end product(s). With respectto the native host cell, it is known that Y. lipolytica can naturallyproduce 18:2 fatty acids and thus possesses a native Δ12 desaturase (SEQID NOs:23 and 24; see WO 2004/104167). With respect to the desired endproducts, the consequences of Δ6 desaturase/Δ6 elongase pathwayexpression as opposed to Δ9 elongase/Δ8 desaturase pathway expressionhave been described above, in terms of the final fatty acid profile ofoil so produced (i.e., % GLA in the final composition of high ARA oil).

In some embodiments, it will therefore be desirable to produce ARA viathe Δ6 desaturase/Δ6 elongase pathway. Thus, at a minimum, the followinggenes must be introduced into the host organism and expressed for ARAbiosynthesis: a Δ6 desaturase, a C_(18/20) elongase and a Δ5 desaturase.In a further preferred embodiment, the host strain additionally includesat least one of the following: a Δ9 desaturase, a Δ12 desaturase, aC_(14/16) elongase and a C_(16/18) elongase.

In alternate embodiments, it is desirable to produce ARA withoutco-synthesis of GLA (thus requiring expression of the Δ9 elongase/Δ8desaturase pathway). This strategy thereby minimally requires thefollowing genes to be introduced into the host organism and expressedfor ARA biosynthesis: a Δ9 elongase, a Δ8 desaturase and a Δ5desaturase. In a further preferred embodiment, the host strainadditionally includes at least one of the following: a Δ9 desaturase, aΔ12 desaturase, a C_(14/16) elongase and a C_(16/18) elongase.

One skilled in the art will be able to identify various candidate genesencoding each of the enzymes desired for ARA biosynthesis. Usefuldesaturase and elongase sequences may be derived from any source, e.g.,isolated from a natural source (from bacteria, algae, fungi, plants,animals, etc.), produced via a semi-synthetic route or synthesized denovo. Although the particular source of the desaturase and elongasegenes introduced into the host is not critical to the invention,considerations for choosing a specific polypeptide having desaturase orelongase activity include: 1.) the substrate specificity of thepolypeptide; 2.) whether the polypeptide or a component thereof is arate-limiting enzyme; 3.) whether the desaturase or elongase isessential for synthesis of a desired PUFA; and/or 4.) co-factorsrequired by the polypeptide. The expressed polypeptide preferably hasparameters compatible with the biochemical environment of its locationin the host cell. For example, the polypeptide may have to compete forsubstrate with other enzymes in the host cell. Analyses of the K_(M) andspecific activity of the polypeptide therefore may be considered indetermining the suitability of a given polypeptide for modifying PUFAproduction in a given host cell. The polypeptide used in a particularhost cell is one that can function under the biochemical conditionspresent in the intended host cell but otherwise can be any polypeptidehaving desaturase or elongase activity capable of modifying the desiredPUFA.

In additional embodiments, it will also be useful to consider theconversion efficiency of each particular desaturase and/or elongase.More specifically, since each enzyme rarely functions with 100%efficiency to convert substrate to product, the final lipid profile ofun-purified oils produced in a host cell will typically be a mixture ofvarious PUFAs consisting of the desired ARA, as well as various upstreamintermediary PUFAs (e.g., as opposed to 100% ARA oil). Thus,consideration of each enzyme's conversion efficiency is also animportant variable when optimizing biosynthesis of ARA, that must beconsidered in light of the final desired lipid profile of the product.

With each of the considerations above in mind, candidate genes havingthe appropriate desaturase and elongase activities can be identifiedaccording to publicly available literature (e.g., GenBank), the patentliterature, and experimental analysis of microorganisms having theability to produce PUFAs. For instance, the following GenBank AccessionNumbers refer to examples of publicly available genes useful in ARAbiosynthesis: AY131238, Y055118, AY055117, AF296076, AF007561, L11421,NM_(—)031344, AF465283, AF465281, AF110510, AF465282, AF419296,AB052086, AJ250735, AF126799, AF126798 (Δ6 desaturases); AF390174 (Δ9elongase); AF139720 (Δ8 desaturase); AF199596, AF226273, AF320509,AB072976, AF489588, AJ510244, AF419297, AF07879, AF067654, AB022097 (Δ5desaturases); AAG36933, AF110509, AB020033, AAL13300, AF417244,AF161219, AY332747, AAG36933, AF110509, AB020033, AAL13300, AF417244,AF161219, X86736, AF240777, AB007640, AB075526, AP002063 (Δ12desaturases); AF338466, AF438199, E11368, E11367, D83185, U90417,AF085500, AY504633, NM_(—)069854, AF230693 (Δ9 desaturases); andNP_(—)012339, NP_(—)009963, NP_(—)013476, NP_(—)599209, BAB69888,AF244356, AAF70417, AAF71789, AF390174, AF428243, NP_(—)955826,AF206662, AF268031, AY591335, AY591336, AY591337, AY591338, AY605098,AY605100, AY630573 (C_(14/16), C_(16/18) and C_(18/20), elongases).Similarly, the patent literature provides many additional DNA sequencesof genes (and/or details concerning several of the genes above and theirmethods of isolation) involved in PUFA production [e.g., WO 02/077213(Δ9 elongases); WO 00/34439 and WO 04/057001 (Δ8 desaturases); U.S. Pat.No. 5,968,809 (Δ6 desaturases); U.S. Pat. No. 5,972,664 and U.S. Pat.No. 6,075,183 (Δ5 desaturases); WO 94/11516, U.S. Pat. No. 5,443,974, WO03/099216 and WO 05/047485 (Δ12 desaturases); WO 91/13972 and U.S. Pat.No. 5,057,419 (Δ9 desaturases); and, WO 00/12720, U.S. Pat. No.6,403,349, U.S. Pat. No. 6,677,145, U.S. 2002/0139974A1, U.S.2004/0111763 (C_(14/16), C_(16/18) and C_(18/20) elongases)]. Each ofthese patents and applications are herein incorporated by reference intheir entirety.

It is contemplated that the examples above are not intended to belimiting and numerous other genes encoding: (1) Δ6 desaturases,C_(18/20) elongases and Δ5 desaturases (and optionally other genesencoding Δ9 desaturases, Δ12 desaturases, C_(14/16) elongases and/orC_(16/18) elongases); or (2) Δ9 elongases, Δ8 desaturases and Δ5desaturases (and optionally other genes encoding Δ9 desaturases, Δ12desaturases, C_(14/16) elongases and/or C_(16/18) elongases) derivedfrom different sources would be suitable for introduction into Yarrowialipolytica.

Preferred Genes for ARA Synthesis

Despite the wide selection of desaturases and elongases that could besuitable for expression in Yarrowia lipolytica, however, in preferredembodiments of the present invention the desaturases and elongases areselected from the following (or derivatives thereof):

TABLE 5 Preferred Desaturases And Elongases For ARA Biosynthesis InYarrowia lipolytica SEQ ID ORF Organism Reference NOs Δ6 MortierellaGenBank Accession 1, 2 desaturase alpina No. AF465281; U.S. Pat. No.5,968,809 Δ6 Mortierella GenBank Accession 4, 5 desaturase alpina No.AB070555 C_(18/20) Mortierella GenBank Accession 17, 18 elongase alpinaNo. AX464731; WO (“ELO1”) 00/12720 C_(18/20) Thraustochytrium U.S. Pat.No. 20, 21 elongase aureum 6,677,145 (“ELO2”) Δ9 elongase IsochrysisGenBank Accession 39, 40 galbana No. AF390174 Δ8 Euglena Co-pending U.S.44, 45 desaturase gracillis patent application No. 11/166993 Δ5Mortierella GenBank Accession 6, 7 desaturase alpina No. AF067654; U.S.Pat. No. 6,075,183 Δ5 Isochrysis WO 02/081668 A2 8, 9 desaturase galbanaΔ5 Homo sapiens GenBank Accession 11, 12 desaturase No. NP_037534C_(16/18) Yarrowia — 61, 62 elongase lipolytica (“YE2”) C_(16/18)Mortierella — 53, 54 elongase alpina (“ELO3”) C_(16/18) Rattus GenBankAccession 50, 51 elongase norvegicus No. AB071986 (rELO2) C_(14/16)Yarrowia — 64, 65 elongase lipolytica (“YE1”) Δ12 Yarrowia WO2004/104167 23, 24 desaturase lipolytica Δ12 Mortieralla GenBankAccession 25, 26 desaturase isabellina No. AF417245 Δ12 Fusarium WO2005/047485 27, 28 desaturase moniliforme (Fm d12) Δ12 AspergillusContig 1.15 (scaffold 1) 29, 30 desaturase nidulans in the A. nidulansgenome (An d12) project; AAG36933; WO 2005/047485 Δ12 AspergillusGenBank Accession  31 desaturase flavus No. AY280867 (VERSIONAY280867.1; gi: 30721844); WO 2005/047485 Δ12 Aspergillus AFA.133c344248:  32 desaturase fumigatus 345586 reverse (Afd12p) (AfA5C5.001c)in the Aspergillus fumigatus genome project; WO 2005/047485 Δ12Magnaporthe Locus MG01985.1 in 33, 34 desaturase grisea contig 2.375 in(Mg d12) the M. grisea genome project; WO 2005/ 047485 Δ12 NeurosporaGenBank Accession 35, 36 desaturase crassa No. AABX01000374; (Nc d12) WO2005/047485 Δ12 Fusarium Contig 1.233 in 37, 38 desaturase gramineariumthe F. graminearium (Fg d12) genome project; WO 2005/047485 Δ12Mortierella GenBank Accession 357, 358 desaturase alpina No. AB020033(Mad12) Δ12 Saccharomyces GenBank Accession 359 desaturase kluyveri No.BAD08375 (Skd12) Δ12 Kluyveromyces gnl|GLV|KLLA0B00473g 360, 361desaturase lactis ORF from KIIa0B: (Kld12p) 35614 . . . 36861 antisense(m) of K. lactis database of the “Yeast project Genolevures” (Center forBioinformatics, LaBRI, Talence Cedex, France) Δ12 Candida GenBankAccession 362 desaturase albicans No. EAK94955 (Cad12p) Δ12 DebaryomycesGenBank Accession 363 desaturase hansenii No. CAG90237 (Dhd12p) CBS767Note: The Aspergillus fumigatus genome project is sponsored by SangerInstitute, collaborators at the University of Manchester and TheInstitute of Genome Research (TIGR); the A. nidulans genome project issponsored by the Center for Genome Research (CGR), Cambridge, MA; the M.grisea genome project is sponsored by the CGR and International RiceBlast Genome Consortium; the F. graminearium genome project is sponsoredby the CGR and the International Gibberella zeae Genomics Consortium(IGGR).

Applicants have analyzed of various elongases, to either determine orconfirm each enzyme's substrate specificity and/or substrate selectivitywhen expressed in Yarrowia lipolytica. For example, although the codingsequences of the two Y. lipolytica elongases were publically availableand each protein was annotated as a putative long-chain fatty-acylelongase or shared significant homology to other fatty acid elongases,the substrate specificity of these enzymes had never been determined.Based on the analyses performed herein, YE1 was positively determined tobe a fatty acid elongase that preferentially used C₁₄ fatty acids assubstrates to produce C₁₆ fatty acids (i.e., a C_(14/16) elongase) andYE2 was determined to be a fatty acid elongase that preferentially usedC₁₆ fatty acids as substrates to produce C₁₈ fatty acids (i.e., aC_(16/18) elongase). Relatedly, upon identification of the novel M.alpina ELO3 gene, the sequence was characterized as homologous to otherfatty acid elongases; however, lipid profile analyses were required toconfirm the specificity of ELO3 as a C_(16/18) elongase.

With respect to Δ12 desaturase, Applicants have made the surprisingdiscovery that the Fusarium moniliforme Δ12 desaturase (encoded by SEQID NO:27) functions with greater efficiency than the native Yarrowialipolytica Δ12 desaturase in producing 18:2 in Y. lipolytica (see WO2005/047485). Specifically, expression of the F. moniliforme Δ12desaturase under the control of the TEF promoter in Y. lipolytica wasdetermined to produce higher levels of 18:2 (68% product accumulation ofLA) than were previously attainable by expression of a chimeric geneencoding the Y. lipolytica Δ12 desaturase under the control of the TEFpromoter (59% product accumulation of LA). This corresponds to adifference in percent substrate conversion (calculated as([18:2+18:3]/[18:1+18:2+18:3])*100) of 85% versus 74%, respectively. Onthe basis of these results, expression of the present fungal F.moniliforme Δ12 desaturase is preferred relative to other known Δ12desaturases as a means to engineer a high ARA-producing strain of Y.lipolytica (however, one skilled in the art would expect that theactivity of the F. moniliforme Δ12 desaturase could be enhanced in Y.lipolytica, following e.g., codon-optimization).

Despite the current identification of the F. moniliforme Δ12 enzyme asthe preferred Δ12 desaturase, five new Δ12 desaturases have recentlybeen identified that could possibly function with improved efficiency inYarrowia lipolytica. Specifically, the Saccharomyces kluyveri Δ12desaturase (GenBank Accession No. BAD08375) was described in Watanabe etal. (Biosci. Biotech. Biocheml. 68(3):721-727 (2004)), while that fromMortierella alpina (GenBank Accession No. AB182163) was described bySakuradani et al. (Eur. J. Biochem. 261(3):812-820 (1999)). Since bothsequences were subsequently utilized to identify S. kluyveri and M.alpina Δ15 desaturases (GenBank Accession No. BAD11952 and No. AB182163,respectively), these two pairs of proteins provided additional examplesof closely related fungal Δ12 and Δ15 desaturases similar to those ofFusarium moniliforme, Aspergillus nidulans, Magnaporthe grisea,Neurospora crassa and Fusarium graminearium (see WO 2005/047480 and WO2005/047485). This finding offered additional support to the Applicants'previous hypothesis that “pairs” of fungal Δ12 desaturase-like sequenceslikely comprise one protein having Δ15 desaturase activity and oneprotein having Δ12 desaturase activity. Similar “pairs” of Δ12desaturase-like proteins were thus identified herein in Kluyveromyceslactis, Candida albicans and Debaryomyces hansenii CBS767; and, aspredicted, one member of each pair aligned more closely to thepreviously identified S. kluyveri Δ12 desaturase (i.e., K. lactisgnl|GLV|KLLA0B00473g ORF, C. albicans GenBank Accession No. EAK94955 andD. hansenii CBS767 GenBank Accession No. CAG90237) while the otheraligned more closely to the S. kluyveri Δ15 desaturase (i.e., K. lactisGenBank Accession No. XM 451551, D. hansenii CBS767 GenBank AccessionNo. CAG88182, C. albicans GenBank Accession No. EAL03493). Thus, basedon this analysis, the Applicants have identified the desaturasesidentified herein as SEQ ID NOs:358, 359, 361, 362 and 363 as putativefungal Δ12 desaturases whose overexpression in Y. lipolytica could beuseful to increase production of ω-6 fatty acids.

In additional embodiments, the Applicants have identified a means toreadily distinguish fungal sequences having Δ12 desaturase activity asopposed to Δ15 desaturase activity. Specifically, when an amino acidsequence alignment was analysed that comprised Δ12 desaturases (i.e.,Mad12, Skd12, Nc d12, Fm d12, Mg d12, An d12, Fg d12, Dhd12p, Kld12p,Cad12p and Afd12p (see Table above)), as well as Δ15 desaturases (i.e.,from Fusarium moniliforme, Aspergillus nidulans, Magnaporthe grisea,Neurospora crassa, F. graminearium, Mortierella alpina, K. lactis, C.albicans, Saccharomyces kluyveri, D. hansenii CBS767 and Aspergillusfumigatus), it became apparent that all of the fungal Δ15 or Δ12desaturases contained either an Ile or Val amino acid residue,respectively, at the position that corresponds to position 102 of theFusarium moniliforme Δ15 desaturase (SEQ ID NO:2 in WO 2005/047479) andthat is only three amino acid residues away from the highly conservedHis Box 1 (“HECGH”; SEQ ID NO:373) (Table 6).

TABLE 6 Amino Acid Alignment Around The Conserved His Box 1 Of FungalΔ12 And Δ15 Desaturases Corresponding Amino Acid Residues Within CodingDesaturase Sequence Motif Desaturase 107-118 of SEQ ID NO: 358

Mad12 116-127 of SEQ ID NO: 359

Skd12 153-164 of SEQ ID NO: 36

Nc d12 149-160 of SEQ ID NO: 28

Fm d12 160-171 of SEQ ID NO: 34

Mg d12 143-154 of SEQ ID NO: 30

An d12 130-141 of SEQ ID NO: 38

Fg d12 106-117 of SEQ ID NO: 361

Kld12p 135-146 of SEQ ID NO: 362

Cad12p 120-131 of SEQ ID NO: 363

Dhd12p 142-153 of SEQ ID NO: 32

Afd12p 105-116 of GenBank Accession No. AB182163

M. alpina Δ15 117-128 of GenBank Accession No. BAD11952

S. kluyveri Δ15 119-130 of SEQ ID NO: 14 in WO 2005/047479

N. crassa Δ15 101-112 of SEQ ID NO: 2 in WO 2005/047479

F. moniliforme Δ15 95-106 of SEQ ID NO: 12 in WO 2005/047479

M. grisea Δ15 88-99 of SEQ ID NO: 6 in WO 2005/047479

A. nidulans Δ15 101-112 of SEQ ID NO: 18 in WO 2005/047479

F. gramin- earium Δ15 117-128 of GenBank Accession No. XM_451551

K. lactis Δ15 130-141 of GenBank Accession No. EAL03493

C. albicans Δ15 132-143 of GenBank Accession No. CAG88182

D. hansenii CBS767 Δ15 94-105 of GenBank Accession No. EAL85733

A. fumigatus Δ15

The Applicants conclude that Ile and Val at this position is adeterminant of Δ15 and Δ12 desaturase specificity, respectively, infungal desaturases. More specifically, the Applicants propose that anyfungal Δ12 desaturase-like protein with Ile at the correspondingresidue(s) (i.e., or the motif IXXHECGH [SEQ ID NO:374]) will be a Δ15desaturase and any fungal Δ12 desaturase-like protein with Val at thecorresponding residue(s) (i.e., or the motif VXXHECGH [SEQ ID NO:375])will be a Δ12 desaturase. Thus, this single leucine/valine amino acidwill be an important residue to consider as future fungal desaturasesare identified and annotated. Furthermore, the Applicants alsohypothesize that mutation(s) that result in a Ile-to-Val change at thisposition will alter enzyme specificity, such as towards Δ12desaturation, in genes encoding fungal Δ12 desaturase-like proteins(e.g., the Fusarium monoliforme desaturase described as SEQ ID NO:2 inWO 2005/047479); and, conversely, those mutations that result in aVal-to-Ile change at this position will alter enzyme specificity, suchas towards Δ15 desaturation.

Of course, in alternate embodiments of the present invention, other DNAswhich are substantially identical to the desaturases and elongasesencoded by SEQ ID NOs:2, 5, 7, 9, 12, 18, 21, 24, 26, 28, 30-32, 34, 36,38, 40, 45, 51, 54, 62, 65, 358, 359 and 361-363 also can be used forproduction of ARA in Yarrowia lipolytica. By “substantially identical”is intended an amino acid sequence or nucleic acid sequence exhibitingin order of increasing preference at least 80%, 90% or 95% homology tothe selected polypeptides, or nucleic acid sequences encoding the aminoacid sequence. For polypeptides, the length of comparison sequencesgenerally is at least 16 amino acids, preferably at least 20 amino acidsor most preferably 35 amino acids. For nucleic acids, the length ofcomparison sequences generally is at least 50 nucleotides, preferably atleast 60 nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides.

Homology typically is measured using sequence analysis software, whereinthe term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc., Madison, Wis.); and 4.)the FASTA program incorporating the Smith-Waterman algorithm (W. R.Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), MeetingDate 1992, 111-20. Suhai, Sandor, Ed. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized. In general, such computer softwarematches similar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications.

In more preferred embodiments, codon-optimized genes encodingdesaturases and elongases that are substantially identical to thosedescribed in SEQ ID NOs:2, 9, 12, 18, 21, 40, 45 and 51 are utilized.Specifically, as is well known to one of skill in the art, theexpression of heterologous genes can be increased by increasing thetranslational efficiency of encoded mRNAs by replacement of codons inthe native gene with those for optimal gene expression in the selectedhost microorganism. Thus, it is frequently useful to modify a portion ofthe codons encoding a particular polypeptide that is to be expressed ina foreign host, such that the modified polypeptide uses codons that arepreferred by the alternate host; and, use of host preferred codons cansubstantially enhance the expression of the foreign gene encoding thepolypeptide.

In general, host preferred codons can be determined within a particularhost species of interest by examining codon usage in proteins(preferably those expressed in the largest amount) and determining whichare used with highest frequency. Then, the coding sequence for apolypeptide of interest (e.g., a desaturase, elongase, acyltransferase)can be synthesized in whole or in part using the codons preferred in thehost species. All (or portions) of the DNA also can be synthesized toremove any destabilizing sequences or regions of secondary structurethat would be present in the transcribed mRNA. And, all (or portions) ofthe DNA also can be synthesized to alter the base composition to onemore preferable in the desired host cell.

Additionally, the nucleotide sequences surrounding the translationalinitiation codon ‘ATG’ have been found to affect expression in yeastcells. If the desired polypeptide is poorly expressed in yeast, thenucleotide sequences of exogenous genes can be modified to include anefficient yeast translation initiation sequence to obtain optimal geneexpression. For expression in yeast, this can be done by site-directedmutagenesis of an inefficiently expressed gene by fusing it in-frame toan endogenous yeast gene, preferably a highly expressed gene.Alternatively, as demonstrated herein for Yarrowia lipolytica, one candetermine the consensus translation initiation sequence in the host andengineer this sequence into heterologous genes for their optimalexpression in the host of interest.

In the present invention, several desaturase and elongase genes fromTable 5 were codon-optimized for expression in Yarrowia lipolytica,based on the host preferences described above. This was possible byfirst determining the Y. lipolytica codon usage profile (see WO04/101757) and identifying those codons that were preferred. Then, forfurther optimization of gene expression in Y. lipolytica, the consensussequence around the ‘ATG’ initiation codon was determined (i.e.,‘MAMMATGNHS’ (SEQ ID NO:356), wherein the nucleic acid degeneracy codeused is as follows: M=A/C; S=C/G; H=A/C/T; and N=A/C/G/T). Table 7,below, compares the activity of native and codon-optimized genes whenexpressed in Y. lipolytica and provides details about eachcodon-optimized gene. % Sub. Conv. is the abbreviation for “percentsubstrate conversion” and Codon-Opt. is an abbreviation for“codon-optimized”.

TABLE 7 Most Preferred Codon-Optimized Desaturases And Elongases For ARABiosynthesis In Yarrowia lipolytica Native Total Bases Codon-Opt. Gene %Modified In Gene % Codon-Opt. Native Gene Sub. Conv. Codon-Opt. GeneSub. Conv. Reference SEQ ID NO M. alpina Δ6 desaturase 30% 152 of 1374bp 42% WO 04/101753 3 (GenBank Accession (corresponding No. AF465281) to144 codons) M. alpina high affinity 30% 94 of 957 bp 47% WO 04/101753 19C_(18/20) elongase (corresponding (GenBank Accession to 85 codons) No.AX464731) T. aureum C_(18/20) 33% 114 of 817 bp 46% — 22 elongase(“ELO2”) (corresponding to 108 codons) S. diclina Δ17 23% 127 of 1077 bp45% Co-Pending 16 desaturase (U.S. (corresponding U.S. patent2003/0196217 A1) to 117 codons) application No. 10/840,478 Isochrysisgalbana — 126 of 789 bp 30% — 41 Δ9 elongase (corresponding to 123codons) Euglena gracillis Δ8 — 207 of 1263 bp 75% Co-pending 48desaturase (corresponding U.S. patent to 192 codons) application No.11/166,993 Isochrysis galbana  7% 203 of 1323 bp 32% — 10 Δ5 desaturase(corresponding to 193 codons) Homo sapiens Δ5 — 227 of 1335 bp 30% — 13desaturase (corresponding (GenBank Accession to 207 codons) No.NP_037534) Rattus norvegicus — 127 of 792 bp 43% — 52 C_(16/18) elongase(corresponding (GenBank Accession to 125 codons) No. AB071986)

In additional alternate embodiments of the invention, other DNAs which,although not substantially identical to the preferred desaturases andelongases presented as SEQ ID NOs:3, 10, 13, 16, 19, 22, 41, 48 and 52also can be used for the purposes herein. For example, DNA sequencesencoding Δ6 desaturase polypeptides that would be useful forintroduction into Yarrowia lipolytica according to the teachings of thepresent invention may be obtained from microorganisms having an abilityto produce GLA or STA. Such microorganisms include, for example, thosebelonging to the genera Mortierella, Conidiobolus, Pythium,Phytophathora, Penicillium, Porphyridium, Coidosporium, Mucor, Fusarium,Aspergillus, Rhodotorula and Entomophthora. Within the genusPorphyridium, of particular interest is P. cruentum. Within the genusMortierella, of particular interest are M. elongata, M. exigua, M.hygrophila, M. ramanniana var. angulispora and M. alpina. Within thegenus Mucor, of particular interest are M. circinelloides and M.javanicus.

Alternatively, a related desaturase that is not substantially identicalto the M. alpina Δ6 desaturase, for example, but which can desaturate afatty acid molecule at carbon 6 from the carboxyl end of the moleculewould also be useful in the present invention as a Δ6 desaturase,assuming the desaturase can still effectively convert LA to GLA and/orALA to STA. As such, related desaturases and elongases can be identified(or created) by their ability to function substantially the same as thedesaturases and elongases disclosed herein.

As suggested above, in another embodiment one skilled in the art couldcreate a fusion protein having e.g., both Δ12 desaturase and Δ6desaturase activities suitable for the purposes herein. This would bepossible by fusing together a Δ12 desaturase and Δ6 desaturase with anadjoining linker. Either the Δ12 desaturase or the Δ6 desaturase couldbe at the N-terminal portion of the fusion protein. Means to design andsynthesize an appropriate linker molecule are readily known by one ofskill in the art; for example, the linker can be a stretch of alanine orlysine amino acids and will not affect the fusion enzyme's activity.

Finally, it is well known in the art that methods for synthesizingsequences and bringing sequences together are well established in theliterature. Thus, in vitro mutagenesis and selection, site-directedmutagenesis, chemical mutagenesis, “gene shuffling” methods or othermeans can be employed to obtain mutations of naturally occurringdesaturase and/or elongase genes. This would permit production of apolypeptide having desaturase or elongase activity, respectively, invivo with more desirable physical and kinetic parameters for functioningin the host cell (e.g., a longer half-life or a higher rate ofproduction of a desired PUFA).

In summary, although sequences of preferred desaturase and elongasegenes are presented that encode PUFA biosynthetic pathway enzymessuitable for ARA production in Yarrowia lipolytica, these genes are notintended to be limiting to the invention herein. Numerous other genesencoding PUFA biosynthetic pathway enzymes that would be suitable forthe purposes herein could be isolated from a variety of sources (e.g., awildtype, codon-optimized, synthetic and/or mutant enzyme havingappropriate desaturase or elongase activity). These alternatedesaturases would be characterized by the ability to: 1.) desaturate afatty acid between the 8^(th) and 9^(th) carbon atom numbered from thecarboxyl-terminal end of the molecule and catalyze the conversion of EDAto DGLA (Δ8 desaturases); 2.) catalyze the conversion of LA to GLA (Δ6desaturases); 3.) catalyze the conversion of DGLA to ARA (Δ5desaturases); 4.) catalyze the conversion of oleic acid to LA (Δ12desaturases); and/or 5.) catalyze the conversion of palmitate topalmitoleic acid and/or stearate to oleic acid (Δ9 desaturases). In likemanner, suitable elongases for the purposes herein are not limited tothose from a specific source; instead, the enzymes having use for thepurposes herein are characterized by their ability to elongate a fattyacid carbon chain by 2 carbons relative to the substrate the elongaseacts upon, to thereby produce a mono- or polyunsaturated fatty acid.More specifically, these elongases would be characterized by the abilityto: 1.) elongate LA to EDA (Δ9 elongases); 2.) elongate a C18 substrateto produce a C20 product (C_(18/20) elongases); 3.) elongate a C14substrate to produce a C16 product (C_(14/16) elongases); and/or 4.)elongate a C16 substrate to produce a C18 product (C_(16/18) elongases).Again, it is important to note that some elongases may be capable ofcatalyzing several elongase reactions, as a result of broad substratespecificity.

Acyltransferases and their Role in the Terminal Step of TAG Biosynthesis

Acyltransferases are intimately involved in the biosynthesis of TAGs.Two comprehensive mini-reviews on TAG biosynthesis in yeast, includingdetails concerning the genes involved and the metabolic intermediatesthat lead to TAG synthesis are: D. Sorger and G. Daum, Appl. Microbiol.Biotechnol. 61:289-299 (2003); and H. Müllner and G. Daum, ActaBiochimica Polonica, 51(2):323-347 (2004). Although the authors of thesereviews clearly summarize the different classes of eukaryoticacyltransferase gene families (infra), they also acknowledge thatregulatory aspects of TAG synthesis and formation of neutral lipids inlipid particles remain far from clear.

Four eukaryotic acyltransferase gene families have been identified whichare involved in acyl-CoA-dependent or independent esterificationreactions leading to neutral lipid synthesis:

-   (1) The acyl-CoA:cholesterol acyltransferase (ACAT) family, EC    2.3.1.26 (commonly known as sterol acyltransferases). This family of    genes includes enzymes responsible for the conversion of acyl-CoA    and sterol to CoA and sterol esters. This family also includes    DGAT1, involved in the terminal step of TAG biosynthesis.-   (2) The lecithin:cholesterol acyltransferase (LCAT) family, EC    2.3.1.43. This family of genes is responsible for the conversion of    phosphatidylcholine and a sterol to a sterol ester and    1-acylglycerophosphocholine. This family also includes the    phospholipid:diacylglycerol acyltransferase (PDAT) enzyme involved    in the transfer of an acyl group from the sn-2 position of a    phospholipid to the sn-3 position of 1,2-diacylglycerol resulting in    TAG biosynthesis.-   (3) The diacylglycerol acyltransferase (DAG AT) family, EC 2.3.1.20.    This family of genes (which includes DGAT2) is involved in the    terminal step of TAG biosynthesis.-   (4) The glycerol-3-phosphate acyltransferase and acyl-CoA    lysophosphatidic acid acyltransferase (GPAT/LPAAT) family. GPAT    (E.C. 2.3.1.15) proteins are responsible for the first step of TAG    biosynthesis, while LPAAT (E.C. 2.3.1.51) enzymes are involved in    the second step of TAG biosynthesis. This family also includes    lysophosphatidylcholine acyltransferase (LPCAT) that catalyzes the    acyl exchange between phospholipid and CoA.    Together, these 4 acyltransferase gene families represent    overlapping biosynthetic systems for neutral lipid formation and    appear to be the result of differential regulation, alternate    localization, and different substrate specifies (H. Milliner and G.    Daum, supra). Each of these four gene families will be discussed    herein based on their importance with respect to metabolic    engineering in Yarrowia lipolytica, to enable synthesis of greater    than 10-30% ARA.

The Functionality of Various Acyltransferases

The interplay between many of these acyltransferases in Yarrowialipolytica is schematically diagrammed in FIG. 3. Focusing initially onthe direct mechanism of TAG biosynthesis, the first step in this processis the esterification of one molecule of acyl-CoA tosn-glycerol-3-phosphate via GPAT to produce lysophosphatidic acid (LPA)(and CoA as a by-product). Then, lysophosphatidic acid is converted tophosphatidic acid (PA) (and CoA as a by-product) by the esterificationof a second molecule of acyl-CoA, a reaction that is catalyzed by LPAAT.Phosphatidic acid phosphatase is then responsible for the removal of aphosphate group from phosphatidic acid to yield 1,2-diacylglycerol(DAG). And, finally a third fatty acid is added to the sn-3 position ofDAG by a DAG AT (e.g., DGAT1, DGAT2 or PDAT) to form TAG.

Historically, DGAT1 was thought to be the only enzyme specificallyinvolved in TAG synthesis, catalyzing the reaction responsible for theconversion of acyl-CoA and DAG to TAG and CoA, wherein an acyl-CoA groupis transferred to DAG to form TAG. DGAT1 was known to be homologous toACATs; however, recent studies have identified a new family of DAG ATenzymes that are unrelated to the ACAT gene family. Thus, nomenclaturenow distinguishes between the DAG AT enzymes that are related to theACAT gene family (DGAT1 family) versus those that are unrelated (DGAT2family) (Lardizabal et al., J. Biol. Chem. 276(42):38862-38869 (2001)).Members of the DGAT2 family have been identified in all major phyla ofeukaryotes (fungi, plants, animals and basal eukaryotes).

Even more recently, Dahlqvist et al. (Proc. Nat. Acad. Sci. (USA)97:6487-6492 (2000)) and Oelkers et al. (J. Biol. Chem. 275:15609-15612(2000)) discovered that TAG synthesis can also occur in the absence ofacyl-CoA, via an acyl-CoA-independent mechanism. Specifically, PDATremoves an acyl group from the sn-2 position of a phosphotidylcholinesubstrate for transfer to DAG to produce TAG. This enzyme isstructurally related to the LCAT family; and although the function ofPDAT is not as well characterized as DGAT2, PDAT has been postulated toplay a major role in removing “unusual” fatty acids from phospholipidsin some oilseed plants (Banas, A. et al., Biochem. Soc. Trans.28(6):703-705 (2000)).

With respect to TAG synthesis in Saccharomyces cerevisiae, threepathways have been described (Sandager, L. et al., J. Biol. Chem.277(8):6478-6482 (2002)). First, TAGs are mainly synthesized from DAGand acyl-CoAs by the activity of DGAT2 (encoded by the DGA1 gene). Morerecently, however, a PDAT (encoded by the LRO1 gene) has also beenidentified. Finally, two acyl-CoA:sterol-acyltransferases (encoded bythe ARE1 and ARE2 genes) are known that utilize acyl-CoAs and sterols toproduce sterol esters (and TAGs in low quantities; see Sandager et al.,Biochem. Soc. Trans. 28(6):700-702 (2000)). Together, PDAT and DGAT2 areresponsible for approximately 95% of oil biosynthesis in S. cerevisiae.

Based on several publicly available sequences encoding DGAT1s, DGAT2s,PDATs and ARE2s (infra), the Applicants isolated and characterized thegenes encoding DGAT1 (SEQ ID NO:81), DGAT2 (SEQ ID NOs:89, 91 and 93[wherein SEQ ID NO:89 contains at least two additional nested ORFs asprovided in SEQ ID NOs:91 and 93; the ORF encoded by SEQ ID NO:93 has ahigh degree of similarity to other known DGAT enzymes and disruption inSEQ ID NO:93 eliminated DGAT function of the native gene, therebyconfirming that the polypeptide of SEQ ID NO:94 has DGATfunctionality]), PDAT (SEQ ID NO:76) and ARE2 (SEQ ID NO:78) in Yarrowialipolytica. In contrast to the model developed in S. cerevisiae, whereinPDAT and DGAT2 are responsible for approximately 95% of oilbiosynthesis, however, it was discovered that the PDAT, DGAT2 and DGAT1of Yarrowia lipolytica are responsible for up to ˜95% of oilbiosynthesis (while ARE2 may additionally be a minor contributor to oilbiosynthesis).

The final acyltransferase enzyme whose function could be important inthe accumulation of ARA in the TAG fraction of Yarrowia lipolytica isLPCAT. As shown in FIG. 3, this enzyme (EC 2.3.1.23) is hypothesized tobe responsible for two-way acyl exchange at the sn-2 position ofsn-phosphatidylcholine to enhance ω-6 and ω-3 PUFA biosynthesis. Thishypothesis is based on the following studies: (1) Stymne S, and A. K.Stobart (Biochem J. 223(2):305-14 (1984)), who hypothesized that LPCATaffected exchange between the acyl-CoA pool and phosphatidylcholine (PC)pool; (2) Domergue, F. et al. (J. Bio. Chem. 278:35115 (2003)), whosuggested that accumulation of GLA at the sn-2 position of PC and theinability to efficiently synthesize ARA in yeast was a result of theelongation step involved in PUFA biosynthesis occurring within theacyl-CoA pool, while Δ5 and Δ6 desaturation steps occurred predominantlyat the sn-2 position of PC; (3) Abbadi, A. et al. (The Plant Cell,16:2734-2748 (2004)), who suggested that LPCAT plays a critical role inthe successful reconstitution of a Δ6 desaturase/Δ6 elongase pathway,based on analysis on the constraints of PUFA accumulation in transgenicoilseed plants; and, (4) WO 2004/076617 A2 (Renz, A. et al.), whoprovided a gene encoding LPCAT from Caenorhabditis elegans (T06E8.1)that substantially improved the efficiency of elongation in agenetically introduced Δ6 desaturase/Δ6 elongase pathway in S.cerevisiae. The inventors concluded that LPCAT allowed efficient andcontinuous exchange of the newly synthesized fatty acids betweenphospholipids and the acyl-CoA pool, since desaturases catalyze theintroduction of double bonds in lipid-coupled fatty acids (sn-2 acyl PC)while elongases exclusively catalyze the elongation of CoA esterifiedfatty acids (acyl-CoAs).

Selection of Heterologous Acyltransferase Genes for ARA Synthesis

Since naturally produced PUFAs in Yarrowia lipolytica are limited to18:2 fatty acids (and less commonly, 18:3 fatty acids), it would belikely that the host organism's native genes encoding GPAT, LPAAT (i.e.,LPAAT1 or LPAAT2), DGAT1, DGAT2, PDAT and LPCAT could have difficultyefficiently synthesizing TAGs comprising fatty acids that were 18:3 andgreater in length (e.g., ARA). Thus, in some cases, a heterologous (or“foreign”) acyltransferase could be preferred over a native enzyme.

Numerous acyltransferase genes have been identified in various organismsand disclosed in the public and patent literature. For instance, thefollowing GenBank Accession Numbers refer to examples of publiclyavailable acyltransferase genes useful in lipid biosynthesis: CQ891256,AY441057, AY360170, AY318749, AY093169, AJ422054, AJ311354, AF251795,Y00771, M77003 (GPATs); Q93841, Q22267, Q99943, O15120, Q9NRZ7, Q9NRZ5,Q9NUQ2, O35083, Q9D1E8, Q924S1, Q59188, Q42670, P26647, P44848, Q9ZJN8,O25903 Q42868, Q42870, P26974, P33333, Q9XFW4, CQ891252, CQ891250,CQ891260, CQ891258, CQ891248, CQ891245, CQ891241, CQ891238, CQ891254,CQ891235 (LPAATs); AY445635, BC003717, NM_(—)010046, NM_(—)053437,NM_(—)174693, AY116586, AY327327, AY327326, AF298815 and AF164434(DGAT1s); and NC_(—)001147 [locus NP_(—)014888], NM_(—)012079,NM_(—)127503, AF051849, AJ238008, NM_(—)026384, NM_(—)010046, AB057816,AY093657, AB062762, AF221132, AF391089, AF391090, AF129003, AF251794 andAF164434 (DGAT2s); P40345, O94680, NP_(—)596330, NP_(—)190069 andAB006704 [gi:2351069] (PDATs). Similarly, the patent literature providesmany additional DNA sequences of genes (and/or details concerningseveral of the genes above and their methods of isolation) involved inTAG production [e.g., U.S. Pat. No. 5,210,189, WO 2003/025165 (GPATs);EP1144649 A2, EP1131438, U.S. Pat. No. 5,968,791, U.S. Pat. No.6,093,568, WO 2000/049156 and WO 2004/087902 (LPAATs); U.S. Pat. No.6,100,077, U.S. Pat. No. 6,552,250, U.S. Pat. No. 6,344,548, US2004/0088759A1 and US 20040078836A1 (DGAT1s); US 2003/124126, WO2001/034814, US 2003/115632, US 2003/0028923 and US 2004/0107459(DGAT2s); WO 2000/060095 (PDATs); and WO 2004/076617 A2 (LPCATs).

It is contemplated that the examples above are not intended to belimiting and numerous other genes encoding DGAT1, DGAT2, PDAT, GPAT,LPCAT and LPAAT derived from different sources would be suitable forintroduction into Yarrowia lipolytica. For example, the Applicants haveidentified novel DGAT1s from Mortierella alpina (SEQ ID NOs:83 and 84),Neurospora crassa (SEQ ID NO:85), Gibberella zeae PH-1 (SEQ ID NO:86),Magnaporthe grisea (SEQ ID NO:87) and Aspergillus nidulans (SEQ IDNO:88); and, a novel DGAT2 (SEQ ID NOs:95 and 96), GPAT (SEQ ID NOs:97and 98), LPAAT1 (SEQ ID NOs:67 and 68) and LPAAT2 (SEQ ID NOs:69 and 70)from Mortierella alpina.

Preferred Acyltransferase Genes for ARA Synthesis

Despite the wide selection of acyltransferases that could be suitablefor expression in Yarrowia lipolytica, however, in preferred embodimentsof the present invention the DGAT1, DGAT2, PDAT, GPAT, LPAAT and LPCATare selected from organisms producing significant amounts of longerchain ω-6 (e.g., ARA) and/or ω-3 (e.g., EPA, DHA) PUFAs. Thus, thefollowing enzymes are especially preferred (or derivatives thereof):

TABLE 8 Preferred Heterologous Acyltransferases For Expression In A HighARA-Producing Strain Of Yarrowia lipolytica SEQ ID ORF OrganismReference NOs DGAT1 Mortierella Co-pending U.S. patent 83, 84 alpinaapplication No. 11/024,544 DGAT2 Mortierella Co-pending U.S. patent 95,96 alpina application No. 11/024,545 GPAT Mortierella — 97, 98 alpinaLPAAT1 Mortierella — 67, 68 alpina LPAAT2 Mortierella Co-pending U.S.patent 69, 70 alpina application No.—60/68,9031 LPCAT CaenorhabditisClone T06E8.1; 80 elegans WO 2004/076617 A2

Although not intended to be limiting in the invention herein, M. alpinawas selected as a preferred source of heterologous acyltransferasessince the native organism is capable of synthesizing ARA atconcentrations greater than 50% of the total fatty acids (TFAs). Insimilar manner, C. elegans can produce up to 20-30% of its TFAs as EPA.

Of course, in alternate embodiments of the present invention, other DNAswhich are substantially identical to the acyltransferases encoded by SEQID NOs:67, 68, 69, 70, 80, 83, 84, 95, 96, 97 and 98 also can be usedfor heterologous expression in Yarrowia lipolytica to facilitate theproduction and accumulation of ARA in the TAG fraction. In morepreferred embodiments, codon-optimized genes encoding acyltransferasesthat are substantially identical to those described in SEQ ID NOs:67-70, 80, 83, 84 and 95-98 are utilized.

General Expression Systems, Cassettes, Vectors and Transformation forExpression of Foreign Genes

Microbial expression systems and expression vectors containingregulatory sequences that direct high-level expression of foreignproteins such as those leading to the high-level production of ARA arewell known to those skilled in the art. Any of these could be used toconstruct chimeric genes encoding the preferred desaturases, elongasesand acyltransferases. These chimeric genes could then be introduced intoYarrowia lipolytica using standard methods of transformation to providehigh-level expression of the encoded enzymes.

Vectors or DNA cassettes useful for the transformation of host cells arewell known in the art. The specific choice of sequences present in theconstruct is dependent upon the desired expression products, the natureof the host cell, and the proposed means of separating transformed cellsversus non-transformed cells. Typically, however, the vector or cassettecontains sequences directing transcription and translation of therelevant gene(s), a selectable marker and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene that controls transcriptional initiation (e.g., apromoter) and a region 3′ of the DNA fragment that controlstranscriptional termination (i.e., a terminator). It is most preferredwhen both control regions are derived from genes from the transformedhost cell, although it is to be understood that such control regionsneed not be derived from the genes native to the specific species chosenas a production host.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other constructs to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct can be experimentally determinedso that all introduced genes are expressed at the necessary levels toprovide for synthesis of the desired products.

Constructs comprising the gene(s) of interest may be introduced into ahost cell by any standard technique. These techniques includetransformation (e.g., lithium acetate transformation [Methods inEnzymology, 194:186-187 (1991)]), protoplast fusion, bolistic impact,electroporation, microinjection, or any other method that introduces thegene(s) of interest into the host cell. More specific teachingsapplicable for Yarrowia lipolytica include U.S. Pat. No. 4,880,741 andNo. 5,071,764 and Chen, D. C. et al. (Appl Microbiol Biotechnol.48(2):232-235 (1997)).

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed” or “recombinant” herein. The transformed host willhave at least one copy of the expression construct and may have two ormore, depending upon whether the gene is integrated into the genome,amplified, or is present on an extrachromosomal element having multiplecopy numbers. The transformed host cell can be identified by variousselection techniques, as described in WO 2004/101757 and WO 2005/003310.

Preferred selection methods for use herein are resistance to kanamycin,hygromycin and the amino glycoside G418, as well as ability to grow onmedia lacking uracil, leucine, lysine, tryptophan or histidine. Inalternate embodiments, 5-fluoroorotic acid (5-fluorouracil-6-carboxylicacid monohydrate; “5-FOA”) is used for selection of yeast Ura⁻ mutants.The compound is toxic to yeast cells that possess a functioning URA3gene encoding orotidine 5′-monophosphate decarboxylase (OMPdecarboxylase); thus, based on this toxicity, 5-FOA is especially usefulfor the selection and identification of Ura⁻ mutant yeast strains(Bartel, P. L. and Fields, S., Yeast 2-Hybrid System, Oxford University:New York, v. 7, pp 109-147, 1997).

An alternate preferred selection method utilized herein relies on adominant, non antibiotic marker for Yarrowia lipolytica based onsulfonylurea resistance. The technique is also generally applicable toother industrial yeast strains that may be haploid, diploid, aneuploidor heterozygous. It is expected to overcome two main limitations to thedevelopment of genetic transformation systems for industrial yeaststrains, wherein: (1) there are almost no naturally auxotrophic strains,and the isolation of spontaneous or induced auxotrophic mutants ishindered by the ploidy of the strains; and, (2) the use of antibioticresistance markers may limit the commercial application of strains dueto restrictions on the release of genetically modified organismscarrying antibiotic resistance genes. Although Puig et al. (J. Agric.Food Chem. 46:1689-1693 (1998)) developed a method to overcome theselimitations based on the genetic engineering of a target strain in orderto make it auxotrophic for uridine and the subsequent use of the URA3marker in order to introduce traits of interest, this strategy wasdeemed too laborious for routine work.

The new sulfonylurea resistance selection marker disclosed herein fortransforming Yarrowia lipolytica does not rely on a foreign gene but ona mutant native gene. Thus, it neither requires auxotrophy nor resultsin auxotrophy and allows transformation of wild type strains. Morespecifically, the marker gene (SEQ ID NO:280) is a nativeacetohydroxyacid synthase (AHAS or acetolactate synthase; E.C. 4.1.3.18)that has a single amino acid change (W497L) that confers sulfonyl ureaherbicide resistance. AHAS is the first common enzyme in the pathway forthe biosynthesis of branched-chain amino acids and it is the target ofthe sulfonylurea and imidazolinone herbicides. W497L mutation, based onwork in Saccharomyces cerevisiae (Falco, S. C., et al., Dev. Ind.Microbiol. 30:187-194 (1989); Duggleby, R. G., et. al. Eur. J. Biochem.270:2895 (2003)) is known. Initial testing determined that Yarrowiacells were not naturally resistant to the herbicide as a result of: 1.)poor or no uptake of the herbicide; 2.) the presence of a nativeherbicide-resistant form of AHAS; and/or 3.) use of aherbicide-inactivating mechanism. This enabled synthesis and use of themutant AHAS gene (SEQ ID NO:280) as a means for selection oftransformants.

An additional method for recycling a selection marker relies onsite-specific recombinase systems. Briefly, the site-specificrecombination system consists of two elements: (1) a recombination sitehaving a characteristic DNA sequence [e.g., LoxP]; and (2) a recombinaseenzyme that binds to the DNA sequence specifically and catalyzesrecombination (i.e., excision) between DNA sequences when two or more ofthe recombination sites are oriented in the same direction at a giveninterval on the same DNA molecule [e.g., Cre]. This methodology hasutility as a means of selection, since it is possible to “recycle” apair of preferred selection markers for their use in multiple sequentialtransformations.

More specifically, an integration construct is created comprising atarget gene that is desirable to insert into the host genome (e.g., adesaturase, elongase, acyltransferase), as well as a first selectionmarker (e.g., Ura3, hygromycin phosphotransferase [HPT]) that is flankedby recombination sites. Following transformation and selection of thetransformants, the first selection marker is excised from the chromosomeby the introduction of a replicating plasmid carrying a second selectionmarker (e.g., sulfonylurea resistance [AHAS]) and a recombinase suitableto recognize the site-specific recombination sites introduced into thegenome. Upon selection of those transformants carrying the second markerand confirmation of excision of the first selection marker from the hostgenome, the replicating plasmid is then cured from the host in theabsence of selection. This produces a transformant that possessesneither the first nor second selection marker, and thus the cured strainis available for another round of transformation. One skilled in the artwill recognize that the methodology is not limited to the particularselection markers or site-specific recombination system described above.

Overexpression of Foreign Genes in Yarrowia lipolytica

As is well known to one of skill in the art, merely inserting a gene(e.g., a desaturase) into a cloning vector does not ensure that it willbe successfully expressed at the level needed. It may be desirable tomanipulate a number of different genetic elements that control aspectsof transcription, translation, protein stability, oxygen limitation andsecretion from the host cell. More specifically, gene expression may becontrolled by altering the following: the nature of the relevanttranscriptional promoter and terminator sequences; the number of copiesof the cloned gene; whether the gene is plasmid-borne or integrated intothe genome of the host cell; the final cellular location of thesynthesized foreign protein; the efficiency of translation in the hostorganism; the intrinsic stability of the cloned gene protein within thehost cell; and the codon usage within the cloned gene, such that itsfrequency approaches the frequency of preferred codon usage of the hostcell. Several of these methods of overexpression will be discussedbelow, and are useful in the present invention as a means to overexpresse.g., desaturases, elongases and acyltransferases in Yarrowialipolytica.

Expression of the desired gene(s) can be increased at thetranscriptional level through the use of a stronger promoter (eitherregulated or constitutive) to cause increased expression, byremoving/deleting destabilizing sequences from either the mRNA or theencoded protein, or by adding stabilizing sequences to the mRNA (U.S.Pat. No. 4,910,141).

Initiation control regions or promoters which are useful to driveexpression of desaturase, elongase and acyltransferase genes in thedesired host cell are numerous and familiar to those skilled in the art.Virtually any promoter capable of directing expression of these genes inYarrowia lipolytica is suitable for the present invention. Expression inthe host cell can be accomplished in a transient or stable fashion.Transient expression can be accomplished by inducing the activity of aregulatable promoter operably linked to the gene of interest;alternatively, stable expression can be achieved by the use of aconstitutive promoter operably linked to the gene of interest. As anexample, when the host cell is yeast, transcriptional and translationalregions functional in yeast cells are provided, particularly from thehost species. The transcriptional initiation regulatory regions can beobtained, for example, from: 1.) genes in the glycolytic pathway, suchas alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase,phosphoglycerate mutase, fructose-bisphosphate aldolase,phosphoglucose-isomerase, phosphoglycerate kinase, glycerol-3-phosphateO-acyltransferase, etc.; or 2.) regulatable genes, such as acidphosphatase, lactase, metallothionein, glucoamylase, the translationelongation factor EF1-α (TEF) protein (U.S. Pat. No. 6,265,185),ribosomal protein S7 (U.S. Pat. No. 6,265,185), ammonium transporterproteins, export proteins, etc. Any one of a number of regulatorysequences can be used, depending upon whether constitutive or inducedtranscription is desired, the efficiency of the promoter in expressingthe ORF of interest, the ease of construction and the like. The examplesprovided above are not intended to be limiting in the invention herein.

As one of skill in the art is aware, a variety of methods are availableto compare the activity of various promoters. This type of comparison isuseful to facilitate a determination of each promoter's strength for usein future applications wherein a suite of promoters would be necessaryto construct chimeric genes useful for the production of ω-6 and ω-3fatty acids. Thus, it may be useful to indirectly quantitate promoteractivity based on reporter gene expression (i.e., the E. coli geneencoding β-glucuronidase (GUS)). In alternate embodiments, it maysometimes be useful to quantify promoter activity using morequantitative means. One suitable method is the use of real-time PCR (fora general review of real-time PCR applications, see Ginzinger, D. J.,Experimental Hematology, 30:503-512 (2002)). Real-time PCR is based onthe detection and quantitation of a fluorescent reporter. This signalincreases in direct proportion to the amount of PCR product in areaction. By recording the amount of fluorescence emission at eachcycle, it is possible to monitor the PCR reaction during exponentialphase where the first significant increase in the amount of PCR productcorrelates to the initial amount of target template. There are twogeneral methods for the quantitative detection of the amplicon: (1) useof fluorescent probes; or (2) use of DNA-binding agents (e.g.,SYBR-green I, ethidium bromide). For relative gene expressioncomparisons, it is necessary to use an endogenous control as an internalreference (e.g., a chromosomally encoded 16S rRNA gene), therebyallowing one to normalize for differences in the amount of total DNAadded to each real-time PCR reaction. Specific methods for real-time PCRare well documented in the art. See, for example, the Real Time PCRSpecial Issue (Methods, 25(4):383-481 (2001)).

Following a real-time PCR reaction, the recorded fluorescence intensityis used to quantitate the amount of template by use of: 1.) an absolutestandard method (wherein a known amount of standard such as in vitrotranslated RNA (cRNA) is used); 2.) a relative standard method (whereinknown amounts of the target nucleic acid are included in the assaydesign in each run); or 3.) a comparative C_(T) method (ΔΔC_(T)) forrelative quantitation of gene expression (wherein the relative amount ofthe target sequence is compared to any of the reference values chosenand the result is given as relative to the reference value). Thecomparative C_(T) method requires one to first determine the difference(ΔC_(T)) between the C_(T) values of the target and the normalizer,wherein: ΔC_(T)=C_(T) (target)−C_(T) (normalizer). This value iscalculated for each sample to be quantitated and one sample must beselected as the reference against which each comparison is made. Thecomparative ΔΔC_(T) calculation involves finding the difference betweeneach sample's ΔC_(T) and the baseline's ΔC_(T), and then transformingthese values into absolute values according to the formula 2^(−ΔΔCT).

Despite the wide selection of promoters that could be suitable forexpression in Yarrowia lipolytica, however, in preferred embodiments ofthe present invention the promoters are selected from those shown belowin Table 9 (or derivatives thereof).

TABLE 9 Native Promoters Preferred For Overexpression In Yarrowialipolytica Promoter Activity SEQ ID Name Location* Native Gene “Rank”Reference NO TEF — translation 1 U.S. Pat. No. 6,265,185 166 elongationfactor (Muller et al.); EF1-α GenBank Accession No. AF054508 GPD −968 bpto +3 glyceraldehyde-3- 2 WO 2005/003310 158 bp phosphate- dehydrogenaseGPM −875 bp to +3 phospho- 1 WO 2005/003310 160 bp glycerate mutase FBA−1001 bp to fructose- 4 WO 2005/049805 161 −1 bp bisphosphate aldolaseFBAIN −804 bp to +169 fructose- 7 WO 2005/049805 162 bp (including abisphosphate 102 bp intron aldolase [+64 to +165]) FBAINm −804 bp to+169 fructose- 5 WO 2005/049805 163 bp with bisphosphate modification*** aldolase GPDIN −973 bp to +201 glyceraldehyde-3- 3 Co-pending U.S.159 bp (including a phosphate- patent application 146 bp introndehydrogenase No. 11/183,664 [+49 to +194]) GPAT −1130 to +3 glycerol-3-5 Co-pending U.S. 164 bp phosphate O- patent application acyltransferaseNo. 11/225,354 YAT1 −778 to −1 ammonium 6 Co-pending U.S. 165 bptransporter patent application enzyme No. 11/185,301 EXP1 −1000 to −1export protein 6 — 364 bp *Location is with respect to the native gene,wherein the ‘A’ position of the ‘ATG’ translation initiation codon isdesignated as +1. *** The FBAINm promoter is a modified version of theFBAIN promoter, wherein FBAINm has a 52 bp deletion between the ATGtranslation initiation codon and the intron of the FBAIN promoter(thereby including only 22 amino acids of the N-terminus) and a newtranslation consensus motif after the intron. Furthermore, while theFBAIN promoter generates a fusion protein when fused with the codingregion of a gene to be expressed, the FBAINm promoter does not generatesuch a fusion protein.

The activity of GPM is about the same as TEF, while the activity of GPD,FBA, FBAIN, FBAINm, GPDIN, GPAT, YAT1 and EXP1 are all greater than TEF(activity is quantified in a relative manner in the column titled“Activity Rank”, wherein a ‘1’ corresponds to the promoter with lowestactivity, while a ‘7’ corresponds to the promoter with highestactivity). This quantitation is based on comparative studies whereineach promoter was used for creation of a chimeric gene possessing the E.coli gene encoding β-glucuronidase (GUS) as a reporter (Jefferson, R. A.Nature. 14; 342:837-838 (1989)) and a ˜100 bp of the 3′ region of theYarrowia Xpr gene. GUS activity in each expressed construct was measuredby histochemical and/or fluorometric assays (Jefferson, R. A. Plant Mol.Biol. Reporter 5:387-405 (1987)) and/or by use of Real Time PCR.

The YAT1 promoter is unique in that it is characterized by theApplicants as the first promoter identified within Yarrowia that isinducible under oleaginous conditions (i.e., nitrogen limitation).Specifically, although the YAT1 promoter is active in media containingnitrogen (e.g., up to about 0.5% ammonium sulfate), the activity of thepromoter increases when the host cell is grown in nitrogen-limitingconditions (e.g., in medium containing very low levels of ammonium, orlacking ammonium). Thus, a preferred medium would be one that containsless than about 0.1% ammonium sulfate, or other suitable ammonium salts.In a more preferred embodiment, the YAT1 promoter is induced when thehost cell is grown in media with a high carbon to nitrogen (i.e., C:N)ratio, such as a high glucose medium (HGM) containing about 8-12%glucose, and about 0.1% or less ammonium sulfate. These conditions arealso sufficient to induce oleaginy in those yeast that are oleaginous(e.g., Yarrowia lipolytica). Based on GUS activity of cell extracts, theactivity of the YAT1 promoter increased by ˜37 fold when cells wereswitched from a minimal medium into HGM and grown for 24 hrs; after 120hrs in HGM, the activity was reduced somewhat but was still 25× higherthan the activity in minimal medium comprising nitrogen (Example 1).

Of course, in alternate embodiments of the present invention, otherpromoters which are derived from any of the promoter regions describedabove in Table 9 also can be used for heterologous expression inYarrowia lipolytica to facilitate the production and accumulation of ARAin the TAG fraction. In particular, modification of the lengths of anyof the promoters described above can result in a mutant promoter havingidentical activity, since the exact boundaries of these regulatorysequences have not been completely defined. In alternate embodiments,the enhancers located within the introns of the FBAIN and GPDINpromoters can be used to create a chimeric promoter having increasedactivity relative to the native Yarrowia promoter (e.g., chimericGPM::FBAIN and GPM::GPDIN promoters (SEQ ID NOs:167 and 168) hadincreased activity relative to the GPM promoter alone, when drivingexpression of the GUS reporter gene in conjunction with a ˜100 bp of the3′ region of the Yarrowia Xpr gene)).

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same anddifferent genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Preferably, thetermination region is derived from a yeast gene, particularlySaccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces.The 3′-regions of mammalian genes encoding γ-interferon and α-2interferon are also known to function in yeast. Termination controlregions may also be derived from various genes native to the preferredhosts. Optionally, a termination site may be unnecessary; however, it ismost preferred if included. Although not intended to be limiting,termination regions useful in the disclosure herein include: ˜100 bp ofthe 3′ region of the Yarrowia lipolytica extracellular protease (XPR;GenBank Accession No. M17741); the acyl-coA oxidase (Aco3: GenBankAccession No. AJ001301 and No. CAA04661; Pox3: GenBank Accession No.XP_(—)503244) terminators; the Pex20 (GenBank Accession No. AF054613)terminator; the Pex16 (GenBank Accession No. U75433) terminator; theLip1 (GenBank Accession No. Z50020) terminator; the Lip2 (GenBankAccession No. AJ012632) terminator; and the 3-oxoacyl-coA thiolase (OCT;GenBank Accession No. X69988) terminator.

Additional copies (i.e., more than one copy) of the desaturase, elongaseand/or acyltransferase genes described above may be introduced intoYarrowia lipolytica to thereby increase ARA production and accumulation.Specifically, additional copies of genes may be cloned within a singleexpression construct; and/or, additional copies of the cloned gene(s)may be introduced into the host cell by increasing the plasmid copynumber or by multiple integration of the cloned gene into the genome(infra). For example, in one embodiment, a strain of Yarrowia lipolytica(i.e., strain Y2214) was engineered to produce greater than 14% ARA bythe introduction and integration into the Yarrowia genome of chimericgenes comprising: 5 copies of a Δ9 elongase, 3 copies of a Δ8desaturase, 4 copies of a Δ5 desaturase, 1 copy of a Δ12 desaturase and1 copy of a C_(16/18) elongase. Similarly, in an alternate embodiment,strain Y2047 of Y. lipolytica was engineered to produce greater than 11%ARA by the introduction and integration into the Yarrowia genome ofchimeric genes comprising: 1 copy of a Δ6 desaturase, 2 copies of aC_(18/20) elongase, 3 copies of a Δ5 desaturase and 1 copy of a Δ12desaturase.

In general, once the DNA that is suitable for expression in anoleaginous yeast has been obtained (e.g., a chimeric gene comprising apromoter, ORF and terminator), it is placed in a plasmid vector capableof autonomous replication in a host cell; or, it is directly integratedinto the genome of the host cell. Integration of expression cassettescan occur randomly within the host genome or can be targeted through theuse of constructs containing regions of homology with the host genomesufficient to target recombination with the host locus. Although notrelied on in the present invention, all or some of the transcriptionaland translational regulatory regions can be provided by the endogenouslocus where constructs are targeted to an endogenous locus.

In the present invention, the preferred method of expressing genes inYarrowia lipolytica is by integration of linear DNA into the genome ofthe host; and, integration into multiple locations within the genome canbe particularly useful when high level expression of genes are desired.Toward this end, it is desirable to identify a sequence within thegenome that is present in multiple copies.

Schmid-Berger et al. (J. Bact., 176(9):2477-2482 (1994)) discovered thefirst retrotransposon-like element Ylt1 in Yarrowia lipolytica. Thisretrotransposon is characterized by the presence of long terminalrepeats (LTRs; each approximately 700 bp in length) called zeta regions.Ylt1 and solo zeta elements were present in a dispersed manner withinthe genome in at least 35 copies/genome and 50-60 copies/genome,respectively; both elements were determined to function as sites ofhomologous recombination. Further, work by Juretzek et al. (Yeast18:97-113 (2001)) demonstrated that gene expression could bedramatically increased by targeting plasmids into the repetitive regionsof the yeast genome (using linear DNA with LTR zeta regions at bothends), as compared to the expression obtained using low-copy plasmidtransformants. Thus, zeta-directed integration can be ideal as a meansto ensure multiple integration of plasmid DNA into Y. lipolytica,thereby permitting high-level gene expression. Unfortunately, however,not all strains of Y. lipolytica possess zeta regions (e.g., the strainidentified as ATCC #20362). When the strain lacks such regions, it isalso possible to integrate plasmid DNA comprising expression cassettesinto alternate loci to reach the desired copy number for the expressioncassette. For example, preferred alternate loci include: the Lys5 genelocus (GenBank Accession No. M34929), the Ura3 locus (GenBank AccessionNo. AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), theAco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus(Pox3: GenBank Accession No. XP_(—)503244; or, Aco3: GenBank AccessionNo. AJ001301), the Δ12 desaturase gene locus (SEQ ID NO:23), the Lip1gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus(GenBank Accession No. AJ012632).

Advantageously, the Ura3 gene can be used repeatedly in combination with5-FOA selection (supra). More specifically, one can first knockout thenative Ura3 gene to produce a strain having a Ura− phenotype, whereinselection occurs based on 5-FOA resistance. Then, a cluster of multiplechimeric genes and a new Ura3 gene could be integrated into a differentlocus of the Yarrowia genome to thereby produce a new strain having aUra+ phenotype. Subsequent integration would produce a new Ura3− strain(again identified using 5-FOA selection), when the introduced Ura3 geneis knocked out. Thus, the Ura3 gene (in combination with 5-FOAselection) can be used as a selection marker in multiple rounds oftransformation and thereby readily permit genetic modifications to beintegrated into the Yarrowia genome in a facile manner.

For some applications, it will be useful to direct the instant proteinsto different cellular compartments (e.g., the acyl-CoA pool versus thephosphatidylcholine pool). For the purposes described herein, ARA may befound as free fatty acids or in esterified forms such as acylglycerols,phospholipids, sulfolipids or glycolipids. It is envisioned that thechimeric genes described above encoding polypeptides that permit ARAbiosynthesis may be further engineered to include appropriateintracellular targeting sequences.

Juretzek et al. (Yeast, 18:97-113 (2001)) note that the stability ofintegrated plasmid copy number in Yarrowia lipolytica is dependent onthe individual transformants, the recipient strain and the targetingplatform used. Thus, the skilled artisan will recognize that multipletransformants must be screened in order to obtain a strain displayingthe desired expression level and pattern. Such screening may beaccomplished by Southern analysis of DNA blots (Southern, J. Mol. Biol.98:503 (1975)), Northern analysis of mRNA expression (Kroczek, J.Chromatogr. Biomed. Appl., 618(1-2):133-145 (1993)), Western analysis ofprotein expression, phenotypic analysis or GC analysis of the PUFAproducts.

In summary, each of the means described above is useful to increase theexpression of a particular gene product (e.g., a desaturase, elongase,acyltransferase) in Yarrowia lipolytica; and, one skilled in the art ofbiotechnology will readily be capable of selecting the most appropriatecombinations of methods to enable high production of ARA.

Pathway Engineering for Increased ARA Production

Although the methodology described above is useful to up-regulate theexpression of individual heterologous genes, the challenge of increasingARA production in Yarrowia lipolytica is much more complex and mayrequire coordinated manipulation of various metabolic pathways.Manipulations in the PUFA biosynthetic pathway will be addressed first,followed by desirable manipulations in the TAG biosynthetic pathway andthe TAG degradation pathway.

As previously described, the construction of a Yarrowia lipolyticastrain producing greater than 5% ARA in the total oil fraction, or morepreferably greater than 10% ARA in the total oil fraction, or even morepreferably greater than 15-20% ARA in the total oil fraction, or mostpreferably greater than 25-30% ARA in the total oil fraction requires atleast the following genes for expression of the Δ6 desaturase/Δ6elongase pathway: a Δ6 desaturase, a C_(18/20) elongase and a Δ5desaturase; or, at least the following genes for expression of the Δ9elongase/Δ8 desaturase pathway: a Δ9 elongase, a Δ8 desaturase and a Δ5desaturase. In either embodiment, however, it may be desirable toadditionally include a Δ9 desaturase, a Δ12 desaturase, a C_(14/16)elongase and/or a C_(16/18) elongase in the host strain.

In some cases, it may prove advantageous to replace the native Yarrowialipolytica Δ12 desaturase with the Fusarium moniliforme Δ12 desaturase,since the latter shows increased percent substrate conversion (WO2005/047485). More specifically, although both Δ12 desaturases catalyzethe conversion of oleic acid to LA, the two enzymes differ in theiroverall specificity (which thereby affects each enzyme's percentsubstrate conversion). The Applicants have determined that the F.moniliforme Δ12 desaturase has a higher loading capacity of LA onto thesn-2 position of a phosphotidylcholine substrate (thereby facilitatingthe subsequent reaction by Δ6 desaturase) than the Y. lipolytica Δ12desaturase. On this basis, overexpression of the F. moniliforme Δ12desaturase in conjunction with a knockout of the Y. lipolytica Δ12desaturase may result in increased product for subsequent conversion toARA.

In some embodiments, it may be useful to regulate the activity of a hostorganism's native DAG ATs to thereby enable manipulation of the percentof PUFAs within the lipids and oils of the Y. lipolytica host.Specifically, since oil biosynthesis is expected to compete withpolyunsaturation during oleaginy, it is possible to reduce or inactivatethe activity of an organism's one or more acyltransferases (e.g., PDATand/or DGAT1 and/or DGAT2), to thereby reduce the overall rate of oilbiosynthesis while concomitantly increasing the percent of PUFAs(relative to the total fatty acids) that are incorporated into the lipidand oil fractions. This results since polyunsaturation is permitted tooccur more efficiently; or, in other words, by down-regulating theactivity of specific DAG ATs, the substrate competition between oilbiosynthesis and polyunsaturation is reduced in favor ofpolyunsaturation during oleaginy.

One skilled in the art will have the skills necessary to elucidate theoptimum level of down-regulation and the means required to achieve suchinhibition. For example, in some preferred embodiments, it may bedesirable to manipulate the activity of a single DAG AT (e.g., create aDGAT1 knockout, while the activity of PDAT and DGAT2 are not altered).In alternate embodiments, the oleaginous organism comprises at total of“n” native DAG ATs and the activity of a total of “n-1” acyltransferasesare modified to result in a reduced rate of oil biosynthesis, while theremaining acyltransferase retains its wildtype activity. And, in somesituations, it may be desirable to manipulate the activity of all of thenative DAG ATs in some preferred oleaginous organisms, to achieve theoptimum rate of oil biosynthesis with respect to the rate ofpolyunsaturation.

In a similar manner, the Applicants hypothesize that expression ofheterologous acyltransferases in conjunction with knockouts of thecorresponding native Yarrowia lipolytica acyltransferase cansignificantly increase the overall ARA that is produced in the hostcells. Specifically, as suggested previously, heterologous GPAT, LPAAT,DGAT1, DGAT2, PDAT and LPCAT acyltransferases that have specificity forthose fatty acids that are C20 and greater could be preferred over thenative enzymes, since naturally produced PUFAs in Y. lipolytica arelimited to 18:2 fatty acids and the native enzymes may not efficientlycatalyze reactions with longer-chain fatty acids. Based on thisconclusion, the Applicants identified the genes encoding GPAT, LPAAT,DGAT1 and DGAT2 in M. alpina and expressed these genes in engineeredYarrowia hosts producing EPA, resulting in increased PUFA biosynthesis(Examples 14-17 herein). Subsequently, the activity of several of thenative acyltransferases (e.g., DGAT1 and DGAT2) in Y. lipolytica werediminished or knocked-out, as a means to reduce substrate competitionbetween the native and heterologous acyltransferase. Similar resultswould be expected in an engineered Yarrowia host producing ARA.

One must also consider manipulation of pathways and global regulatorsthat affect ARA production. For example, it is useful to increase theflow of carbon into the PUFA biosynthetic pathway by increasing theavailability of the precursors of longer chain saturated and unsaturatedfatty acids, such as palmitate (16:0) and stearic acid (18:0). Thesynthesis of the former is dependent on the activity of a C_(14/16)elongase, while the synthesis of the latter is dependent on the activityof a C_(16/18) elongase. Thus, over-expression of the native Yarrowialipolytica C_(14/16) elongase (SEQ ID NOs:64 and 65) substantiallyincreased the production of 16:0 and 16:1 fatty acids (22% increaserelative to control strains); similarly, over-expression of the nativeY. lipolytica C_(16/18) elongase (SEQ ID NOs:61 and 62) substantiallyincreased the production of 18:0, 18:1, 18:2 and 18:3 fatty acids (18%increase relative to control strains) and reduced the accumulation ofC₁₆ fatty acids (22% decrease relative to control strains). Of course,as demonstrated herein and as suggested by the work of Inagaki, K. etal. (Biosci. Biotech. Biochem. 66(3):613-621 (2002)), in someembodiments of the present invention it may be useful to co-express aheterologous C_(16/18) elongase (e.g., from Rattus norvegicus [GenBankAccession No. AB071986; SEQ ID NOs:50 and 51 herein] and/or from M.alpina [SEQ ID NO:53 and 54. Thus, although a Y. lipolytica host strainmust minimally be manipulated to express either a Δ6 desaturase, aC_(18/20) elongase and a Δ5 desaturase or a Δ9 elongase, a Δ8 desaturaseand a Δ5 desaturase for ARA biosynthesis, in further preferredembodiments the host strain additionally includes at least one of thefollowing: a Δ9 desaturase, a Δ12 desaturase, a C_(14/16) elongaseand/or a C_(16/18) elongase.

In another preferred embodiment, those pathways that affect fatty aciddegradation and TAG degradation can be modified in the Yarrowialipolytica of the present invention, to minimize the degradation of ARAthat accumulates in the cells in either the acyl-CoA pool or in the TAGfraction. These pathways are represented by the acyl-CoA oxidase andlipase genes, respectively. More specifically, the acyl-CoA oxidases (EC1.3.3.6) catalyze a peroxisomal β-oxidation reaction wherein each cycleof degradation yields an acetyl-CoA molecule and a fatty acid that istwo carbon atoms shorter than the fatty acid substrate. Five acyl-CoAoxidase isozymes are present in Yarrowia lipolytica, encoded by thePDX1, PDX2, PDX3, PDX4 and PDX5 genes (also known as the Aco1, Aco2,Aco3, Aco4 and Aco5 genes), corresponding to GenBank Accession Nos.AJ001299-AJ001303, respectively (see also corresponding GenBankAccession Nos. XP_(—)504703, XP_(—)505264, XP_(—)503244, XP_(—)504475and XP_(—)502199). Each of the isozymes has a different substratespecificity; for example, the PDX3 gene encodes an acyl-CoA oxidase thatis active against short-chain fatty acids, whereas the PDX2 gene encodesan acyl-CoA oxidase that is active against longer-chain fatty acids(Wang H. J., et al. J. Bacteriol., 181:5140-5148 (1999)). It iscontemplated that the activity of any one of these genes could bereduced or eliminated, to thereby modify peroxisomal β-oxidation in thehost cell of the invention in a manner that could be advantageous to thepurposes herein. Finally, to avoid any confusion, the Applicants willrefer to the acyl-CoA oxidases as described above as POX genes, althoughthis terminology can be used interchangeably with the Aco genenomenclature, according to some publicly available literature.

Similarly, several lipases (EC 3.1.1.3) have been detected in Y.lipolytica, including intracellular, membrane-bound and extracellularenzymes (Choupina, A., et al. Curr. Genet. 35:297 (1999); Pignede, G.,et al. J. Bacteriol. 182:2802-2810 (2000)). For example, Lip1 (GenBankAccession No. Z50020) and Lip3 (GenBank Accession No. AJ249751) areintracellular or membrane bound, while Lip2 (GenBank Accession No.AJ012632) encodes an extracellular lipase. Each of these lipases aretargets for disruption, since the enzymes catalyze the reaction whereinTAG and water are degraded directly to DAG and a fatty acid anion.

In a further alternate embodiment, the activity of severalphospholipases can be manipulated in the preferred host strain ofYarrowia lipolytica. Phospholipases play a critical role in thebiosynthesis and degradation of membrane lipids. More specifically, theterm “phospholipase” refers to a heterogeneous group of enzymes thatshare the ability to hydrolyze one or more ester linkage inglycerophospholipids. Although all phospholipases target phospholipidsas substrates, each enzyme has the ability to cleave a specific esterbond. Thus, phospholipase nomenclature differentiates individualphospholipases and indicates the specific bond targeted in thephospholipid molecule. For example, phospholipase A₁ (PLA₁) hydrolyzesthe fatty acyl ester bond at the sn-1 position of the glycerol moiety,while phospholipase A₂ (PLA₂) removes the fatty acid at the sn-2position of this molecule. The action of PLA₁ (EC 3.1.1.32) and PLA₂ (EC3.1.1.4) results in the accumulation of free fatty acids and 2-acyllysophospholipid or 1-acyl lysophospholipid, respectively. PhospholipaseC (PLC) (EC 3.1.4.3) hydrolyzes the phosphodiester bond in thephospholipid backbone to yield 1,2-DAG and, depending on the specificphospholipid species involved, phosphatidylcholine,phosphatidylethanolamine, etc. (e.g., PLC₁ is responsible for thereaction: 1-phosphatidyl-1D-myo-inositol4,5-bisphosphate+H₂O=1D-myo-inositol 1,4,5-trisphosphate+DAG; ISC1encodes an inositol phosphosphingolipid-specific phospholipase C [Sawai,H., et al. J. Biol. Chem. 275:39793-39798 (2000)]). The secondphosphodiester bond is cleaved by phospholipase D (PLD) (EC 3.1.4.4) toyield phosphatidic acid and choline or ethanolamine, again depending onthe phospholipid class involved. Phospholipase B (PLB) has thecapability of removing both sn-1 and sn-2 fatty acids and is unique inhaving both hydrolase (wherein the enzyme cleaves fatty acids from bothphospholipids [PLB activity] and lysophospholipids [lysophospholipaseactivity] for fatty acid release) and lysophospholipase-transacylaseactivities (wherein the enzyme can produce phospholipid by transferringa free fatty acid to a lysophospholipid). It may be useful tooverexpress one or more of these phospholipases, in order to increasethe concentration of ARA that accumulates in the total oil fraction ofthe transformant Yarrowia host cells. It is hypothesized that thisresult will be observed because the phospholipases release acyl groupsfrom PC into the CoA pool either for elongation or incorporation intotriglycerides.

In another alternate embodiment, those enzymes in the CDP-cholinepathway responsible for phosphatidylcholine (PC) biosynthesis can alsobe manipulated in the preferred host strain of Yarrowia lipolytica, as ameans to increase overall ARA biosynthesis. The utility of thistechnique has been demonstrated by the overexpression of the Y.lipolytica CPT1 gene encoding diacylglycerol cholinephosphotransferase(EC 2.7.8.2), thereby resulting in increased EPA biosynthesis in anengineered strain of Y. lipolytica. One skilled in the art will befamiliar with the PC biosynthetic pathway and recognize otherappropriate candidate enzymes.

Although methods for manipulating biochemical pathways such as thosedescribed above are well known to those skilled in the art, an overviewof some techniques for reducing or eliminating the activity of a nativegene will be briefly presented below. These techniques would be usefulto down-regulate the activity of the native Yarrowia lipolytica Δ12desaturase, GPAT, LPAAT, DGAT1, DGAT2, PDAT, LPCAT, acyl-CoA oxidase 2(Aco2 or Pox2), acyl-CoA oxidase 3 (Aco3 or Pox3) and/or lipase genes,as discussed above.

Although one skilled in the art will be well equipped to ascertain themost appropriate technique to be utilized to reduce or eliminate theactivity of a native gene, in general, the endogenous activity of aparticular gene can be reduced or eliminated by, for example: 1.)disrupting the gene through insertion, substitution and/or deletion ofall or part of the target gene; 2.) providing a cassette fortranscription of antisense sequences to the gene's transcriptionproduct; 3.) using a host cell which naturally has [or has been mutatedto have] little or none of the specific gene's activity; 4.)over-expressing a mutagenized hereosubunit (i.e., in an enzyme thatcomprises two or more hereosubunits), to thereby reduce the enzyme'sactivity as a result of the “dominant negative effect”; and 5.) usingiRNA technology. In some cases, inhibition of undesired gene pathwayscan also be accomplished through the use of specific inhibitors (e.g.,desaturase inhibitors such as those described in U.S. Pat. No.4,778,630).

For gene disruption, a foreign DNA fragment (typically a selectablemarker gene, but optionally a chimeric gene or chimeric gene clusterconveying a desirable phenotype upon expression) is inserted into thestructural gene to be disrupted in order to interrupt its codingsequence and thereby functionally inactivate the gene. Transformation ofthe disruption cassette into the host cell results in replacement of thefunctional native gene by homologous recombination with thenon-functional disrupted gene (see, for example: Hamilton et al., J.Bacteriol. 171:4617-4622 (1989); Balbas et al., Gene, 136:211-213(1993); Gueldener et al., Nucleic Acids Res. 24:2519-2524 (1996); andSmith et al., Methods Mol. Cell. Biol. 5:270-277 (1996)).

Antisense technology is another method of down-regulating genes when thesequence of the target gene is known. To accomplish this, a nucleic acidsegment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed.This construct is then introduced into the host cell and the antisensestrand of RNA is produced. Antisense RNA inhibits gene expression bypreventing the accumulation of mRNA that encodes the protein ofinterest. The person skilled in the art will know that specialconsiderations are associated with the use of antisense technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of antisense genes may require the use of differentchimeric genes utilizing different regulatory elements known to theskilled artisan.

Although targeted gene disruption and antisense technology offereffective means of down-regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence-based (e.g., mutagenesis via UV radiation/chemical agents oruse of transposable elements/transposons; see WO 04/101757).

In alternate embodiments, the endogenous activity of a particular genecan be reduced by manipulating the regulatory sequences controlling theexpression of the protein. As is well known in the art, the regulatorysequences associated with a coding sequence include transcriptional andtranslational “control” nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of the coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Thus, manipulation of a particular gene's regulatory sequencesmay refer to manipulation of the gene's promoters, translation leadersequences, introns, enhancers, initiation control regions,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures. Thus, for example, the promoterof a DAG AT could be deleted or disrupted, in order to down-regulate theDAG AT's expression and thereby achieve a reduced rate of lipid and oilbiosynthesis. Alternatively, the native promoter driving expression of aDAG AT could be substituted with a heterologous promoter havingdiminished promoter activity with respect to the native promoter.Methods useful for manipulating regulatory sequences are well known tothose skilled in the art.

In summary, using the teachings provided herein, transformant oleaginousmicrobial hosts will produce at least about 5% ARA in the total lipids,preferably at least about 10% ARA in the total lipids, more preferablyat least about 15% ARA in the total lipids, more preferably at leastabout 20% ARA in the total lipids and most preferably at least about25-30% ARA in the total lipids.

Fermentation Processes for ARA Production

The transformed microbial host cell is grown under conditions thatoptimize expression of chimeric genes (e.g., encoding desaturases,elongases, acyltransferases, etc.) and produce the greatest and the mosteconomical yield of ARA. In general, media conditions that may beoptimized include the type and amount of carbon source, the type andamount of nitrogen source, the carbon-to-nitrogen ratio, the oxygenlevel, growth temperature, pH, length of the biomass production phase,length of the oil accumulation phase and the time of cell harvest.Yarrowia lipolytica are generally grown in complex media (e.g., yeastextract-peptone-dextrose broth (YPD)) or a defined minimal media thatlacks a component necessary for growth and thereby forces selection ofthe desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources may include, but are not limitedto: monosaccharides (e.g., glucose, fructose), disaccharides (e.g.,lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch,cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) ormixtures from renewable feedstocks (e.g., cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt). Additionally,carbon sources may include alkanes, fatty acids, esters of fatty acids,monoglycerides, diglycerides, triglycerides, phospholipids and variouscommercial sources of fatty acids including vegetable oils (e.g.,soybean oil) and animal fats. Additionally, the carbon source mayinclude one-carbon sources (e.g., carbon dioxide, methanol,formaldehyde, formate and carbon-containing amines) for which metabolicconversion into key biochemical intermediates has been demonstrated.Hence it is contemplated that the source of carbon utilized in thepresent invention may encompass a wide variety of carbon-containingsources. Although all of the above mentioned carbon sources and mixturesthereof are expected to be suitable in the present invention, preferredcarbon sources are sugars and/or fatty acids. Most preferred is glucoseand/or fatty acids containing between 10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic(e.g., urea or glutamate) source. In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the oleaginousyeast and promotion of the enzymatic pathways necessary for ARAproduction. Particular attention is given to several metal ions (e.g.,Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids and PUFAs(Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R.Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of Yarrowia lipolytica willbe known by one skilled in the art of microbiology or fermentationscience. A suitable pH range for the fermentation is typically betweenabout pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 is preferred as therange for the initial growth conditions. The fermentation may beconducted under aerobic or anaerobic conditions, wherein microaerobicconditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of ARA in Yarrowia lipolytica. This approach is described inWO 2004/101757, as are various suitable fermentation process designs(i.e., batch, fed-batch and continuous) and considerations duringgrowth.

Purification and Processing of ARA

PUFAs, including ARA, may be found in the host microorganism as freefatty acids or in esterified forms such as acylglycerols, phospholipids,sulfolipids or glycolipids, and may be extracted from the host cellthrough a variety of means well-known in the art. One review ofextraction techniques, quality analysis and acceptability standards foryeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology,12(5/6):463-491 (1992)). A brief review of downstream processing is alsoavailable by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312(1997)).

In general, means for the purification of ARA and other PUFAs mayinclude extraction with organic solvents, sonication, supercriticalfluid extraction (e.g., using carbon dioxide), saponification andphysical means such as presses, or combinations thereof. One is referredto the teachings of WO 2004/101757 for additional details.

Oils containing ARA that have been refined and/or purified can behydrogenated, to thereby result in fats with various melting propertiesand textures. Many processed fats, including spreads, confectionaryfats, hard butters, margarines, baking shortenings, etc., requirevarying degrees of solidity at room temperature and can only be producedthrough alteration of the source oil's physical properties. This is mostcommonly achieved through catalytic hydrogenation.

Hydrogenation is a chemical reaction in which hydrogen is added to theunsaturated fatty acid double bonds with the aid of a catalyst such asnickel. Hydrogenation has two primary effects. First, the oxidativestability of the oil is increased as a result of the reduction of theunsaturated fatty acid content. Second, the physical properties of theoil are changed because the fatty acid modifications increase themelting point resulting in a semi-liquid or solid fat at roomtemperature.

There are many variables which affect the hydrogenation reaction andwhich, in turn, alter the composition of the final product. Operatingconditions including pressure, temperature, catalyst type andconcentration, agitation and reactor design are among the more importantparameters which can be controlled. Selective hydrogenation conditionscan be used to hydrogenate the more unsaturated fatty acids inpreference to the less unsaturated ones. Very light or brushhydrogenation is often employed to increase stability of liquid oils.Further hydrogenation converts a liquid oil to a physically solid fat.The degree of hydrogenation depends on the desired performance andmelting characteristics designed for the particular end product. Liquidshortenings, used in the manufacture of baking products, solid fats andshortenings used for commercial frying and roasting operations, and basestocks for margarine manufacture are among the myriad of possible oiland fat products achieved through hydrogenation. A more detaileddescription of hydrogenation and hydrogenated products can be found inPatterson, H. B. W., Hydrogenation of Fats and Oils: Theory andPractice. The American Oil Chemists' Society, 1994.

ARA-Producing Strains of Y. lipolytica for Use in Foodstuffs

The market place currently supports a large variety of food and feedproducts, incorporating ω-3 and/or ω-6 fatty acids (particularly ARA,EPA and DHA). It is contemplated that the yeast microbial oils of theinvention comprising ARA will function in food and feed products toimpart the health benefits of current formulations.

Microbial oils containing ω-3 and/or ω-6 fatty acids produced by theyeast hosts described herein will be suitable for use in a variety offood and feed products including, but not limited to food analogs, meatproducts, cereal products, baked foods, snack foods, and a dairyproducts. Additionally the present microbial oils may be used informulations to impart health benefit in medical foods including medicalnutritionals, dietary supplements, infant formula as well aspharmaceutical products. One of skill in the art of food processing andfood formulation will understand how the amount and composition of themicrobial oil may be added to the food or feed product. Such an amountwill be referred to herein as an “effective” amount and will depend onthe food or feed product, the diet that the product is intended tosupplement or the medical condition that the medical food or medicalnutritional is intended to correct or treat.

Food analogs can be made using processes well known to those skilled inthe art. There can be mentioned meat analogs, cheese analogs, milkanalogs and the like. Meat analogs made from soybeans contain soyprotein or tofu and other ingredients mixed together to simulate variouskinds of meats. These meat alternatives are sold as frozen, canned ordried foods. Usually, they can be used the same way as the foods theyreplace. Examples of meat analogs include, but are not limited to: hamanalogs, sausage analogs, bacon analogs, and the like.

Food analogs can be classified as imitation or substitutes depending ontheir functional and compositional characteristics. For example, animitation cheese need only resemble the cheese it is designed toreplace. However, a product can generally be called a substitute cheeseonly if it is nutritionally equivalent to the cheese it is replacing andmeets the minimum compositional requirements for that cheese. Thus,substitute cheese will often have higher protein levels than imitationcheeses and be fortified with vitamins and minerals.

Milk analogs or nondairy food products include, but are not limited to:imitation milks and nondairy frozen desserts (e.g., those made fromsoybeans and/or soy protein products).

Meat products encompass a broad variety of products. In the UnitedStates “meat” includes “red meats” produced from cattle, hogs and sheep.In addition to the red meats there are poultry items which includechickens, turkeys, geese, guineas, ducks and the fish and shellfish.There is a wide assortment of seasoned and processed meat products:fresh, cured and fried, and cured and cooked. Sausages and hot dogs areexamples of processed meat products. Thus, the term “meat products” asused herein includes, but is not limited to, processed meat products.

A cereal food product is a food product derived from the processing of acereal grain. A cereal grain includes any plant from the grass familythat yields an edible grain (seed). The most popular grains are barley,corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat andwild rice. Examples of a cereal food product include, but are notlimited to: whole grain, crushed grain, grits, flour, bran, germ,breakfast cereals, extruded foods, pastas, and the like.

A baked goods product comprises any of the cereal food productsmentioned above and has been baked or processed in a manner comparableto baking, i.e., to dry or harden by subjecting to heat. Examples of abaked good product include, but are not limited to: bread, cakes,doughnuts, bars, pastas, bread crumbs, baked snacks, mini-biscuits,mini-crackers, mini-cookies, and mini-pretzels. As was mentioned above,oils of the invention can be used as an ingredient.

A snack food product comprises any of the above or below described foodproducts.

A fried food product comprises any of the above or below described foodproducts that has been fried.

The beverage can be in a liquid or in a dry powdered form.

For example, there can be mentioned non-carbonated drinks; fruit juices,fresh, frozen, canned or concentrate; flavored or plain milk drinks,etc. Adult and infant nutritional formulas are well known in the art andcommercially available (e.g., Similac®, Ensure®, Jevity®, and Alimentum®from Ross Products Division, Abbott Laboratories). Infant formulas areliquids or reconstituted powders fed to infants and young children. Theyserve as substitutes for human milk. Infant formulas have a special roleto play in the diets of infants because they are often the only sourceof nutrients for infants. Although breast-feeding is still the bestnourishment for infants, infant formula is a close enough second thatbabies not only survive but thrive. Infant formula is becoming more andmore increasingly close to breast milk.

A dairy product is a product derived from milk. A milk analog ornondairy product is derived from a source other than milk, for example,soymilk as was discussed above. These products include, but are notlimited to: whole milk, skim milk, fermented milk products such asyogurt or sour milk, cream, butter, condensed milk, dehydrated milk,coffee whitener, coffee creamer, ice cream, cheese, etc.

Additional food products into which the ARA-containing oils of theinvention could be included are, for example: chewing gums, confectionsand frostings, gelatins and puddings, hard and soft candies, jams andjellies, white granulated sugar, sugar substitutes, sweet sauces,toppings and syrups, and dry-blended powder mixes.

Health Food Products, and Pharmaceuticals

A health food product is any food product that imparts a health benefitand include functional foods, medical foods, medical nutritionals anddietary supplements. Additionally, microbial oils of the invention maybe used in standard pharmaceutical compositions. The present engineeredstrains of Yarrowia lipolytica or the microbial oils produced therefromcomprising ARA could readily be incorporated into the any of the abovementioned food products, to thereby produce e.g., a functional ormedical food. For example more concentrated formulations comprising ARAinclude capsules, powders, tablets, softgels, gelcaps, liquidconcentrates and emulsions which can be used as a dietary supplement inhumans or animals other than humans.

Use in Dietary Supplements

More concentrated formulations comprising ARA include capsules, powders,tablets, softgels, gelcaps, liquid concentrates and emulsions which canbe used as a dietary supplement in humans or animals other than humans.In particular, the ARA-oil of the present invention is particularlysuitable for incorporation into dietary supplements such as infantformulas or baby food.

Infant formulas are liquids or reconstituted powders fed to infants andyoung children. “Infant formula” is defined herein as an enteralnutritional product which can be substituted for human breast milk infeeding infants and typically is composed of a desired percentage of fatmixed with desired percentages of carbohydrates and proteins in anaqueous solution (e.g., see U.S. Pat. No. 4,670,285). Based on theworldwide composition studies, as well as levels specified by expertgroups, average human breast milk typically contains about 0.20% to0.40% of total fatty acids (assuming about 50% of calories from fat);and, generally the ratio of DHA to ARA would range from about 1:1 to 1:2(see, e.g., formulations of Enfamil LIPIL™ [Mead Johnson & Company] andSimilac Advance™[Ross Products Division, Abbott Laboratories]). Infantformulas have a special role to play in the diets of infants becausethey are often the only source of nutrients for infants; and, althoughbreast-feeding is still the best nourishment for infants, infant formulais a close enough second that babies not only survive but thrive.

Use in Animal Feeds

Animal feeds are generically defined herein as products intended for useas feed or for mixing in feed for animals other than humans. And, as wasmentioned above, the ARA-comprising oils of the invention can be used asan ingredient in various animal feeds.

More specifically, although not limited therein, it is expected that theoils of the invention can be used within pet food products, ruminant andpoultry food products and aquacultural food products. Pet food productsare those products intended to be fed to a pet [e.g., a dog, cat, bird,reptile, rodent]; these products can include the cereal and health foodproducts above, as well as meat and meat byproducts, soy proteinproducts, grass and hay products (e.g., alfalfa, timothy, oat or bromegrass, vegetables). Ruminant and poultry food products are those whereinthe product is intended to be fed to e.g., turkeys, chickens, cattle andswine. As with the pet foods above, these products can include cerealand health food products, soy protein products, meat and meatbyproducts, and grass and hay products as listed above. And,aquacultural food products (or “aquafeeds”) are those products intendedto be used in aquafarming which concerns the propagation, cultivation orfarming of aquatic organisms and/or animals in fresh or marine waters.

It is contemplated that the present engineered strains of Yarrowialipolytica that are producing high concentrations of ARA, EPA and/or DHAwill be especially useful to include in most animal feed formulations.In addition to providing necessary ω-3 and/or ω-6 PUFAs, the yeastitself is a useful source of protein and other feed nutrients (e.g.,vitamins, minerals, nucleic acids, complex carbohydrates, etc.) that cancontribute to overall animal health and nutrition, as well as increase aformulation's palatablility. More specifically, Yarrowia lipolytica(ATCC #20362) has the following approximate chemical composition, as apercent relative to the dry cell weight: 35% protein, 40% lipid, 10%carbohydrate, 5% nucleic acids, 5% ash and 5% moisture. Furthermore,within the carbohydrate fraction, β-glucans comprise approximately 45.6mg/g, mannans comprise approximately 11.4 mg/g, and chitin comprisesapproximately 52.6 mg/g (while trehalose is a minor component[approximately 0.7 mg/g]).

A considerable body of literature has examined the immuno-modulatingeffects of β-glucans, mannans and chitin. The means by which β-glucans,the primary constituents of bacterial and fungal cell walls, stimulatenon-specific immunity (i.e., “immunostimulant effects”) to therebyimprove health of aquaculture species, pets and farm animals and humansare best studied, although both chitin and mannans are similarlyrecognized as useful immunostimulants. Simplistically, an overallenhancement of immune response can be achieved by the use of β-glucans,since these β-1,3-D-polyglucose molecules stimulate the production ofwhite blood cells (e.g., macrophages, neutrophils and monocytes) in anon-specific manner to thereby enable increased sensitivity and defenseagainst a variety of pathogenic antigens or environmental stressors.More specifically, numerous studies have demonstrated that β-glucans:convey enhanced protection against viral, bacterial, fungal andparasitic infections; exert an adjuvant effect when used in conjunctionwith antibiotics and vaccines; enhance wound healing; counter damageresulting from free radicals; enhance tumor regression; modulatetoxicity of bacterial endotoxins; and strengthen mucosal immunity(reviewed in Raa, J. et al., Norwegian Beta Glucan Research, ClinicalApplications of Natural Medicine. Immune: Depressions Dysfunction &Deficiency (1990)). A sample of current literature documenting theutility of yeast β-glucans, mannans and chitins in both traditionalanimal husbandry and within the aquacultural sector include: L. A. Whiteet al. (J. Anim. Sci. 80:2619-2628 (2002)), supplementation in weanlingpigs; K. S. Swanson et al. (J. Nutr. 132:980-989 (2002)),supplementation in dogs; J. Ortuño et al. (Vet. Immunol. Immonopath.85:41-50 (2002)), whole Saccharomyces cerevisiae administered togilthead seabream; A. Rodríguez et al. (Fish Shell. Immuno. 16:241-249(2004)), whole Mucor circinelloides administered to gilthead seabream;M. Bagni et al. (Fish Shell. Immuno. 18:311-325 (2005)), supplementationof sea bass with a yeast extract containing β-glucans; J. Raa (In:Cruz-Suaŕez, L. E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa,M. A. y Civera-Cerecedo, R., (Eds.). Avances en Nutrición Acuícola V.Memorias del V Simposium Internacional de Nutrición Acuícola. 19-22Nov., 2000. Mérida, Yucatán, Mexico), a review of the use ofimmune-stimulants in fish and shellfish feeds.

Based on the unique protein:lipid:carbohydrate composition of Yarrowialipolytica, as well as unique complex carbohydrate profile (comprisingan approximate 1:4:4.6 ratio of mannan:β-glucans:chitin), it iscontemplated that the genetically engineered yeast cells of the presentinvention (or portions thereof) would be a useful additive to animalfeed formulations (e.g., as whole [lyophilized] yeast cells, as purifiedcells walls, as purified yeast carbohydrates or within various otherfractionated forms).

With respect to the aquaculture industry, an increased understanding ofthe nutritional requirements for various fish species and technologicaladvances in feed manufacturing have allowed the development and use ofmanufactured or artificial diets (formulated feeds) to supplement or toreplace natural feeds in the aquaculture industry. In general, however,the general proportions of various nutrients included in aquaculturefeeds for fish include (with respect to the percent by dry diet): 32-45%proteins, 4-28% fat (of which at least 1-2% are ω-3 and/or ω-6 PUFAs),10-30% carbohydrates, 1.0-2.5% minerals and 1.0-2.5% vitamins. A varietyof other ingredients may optionally be added to the formulation. Theseinclude: (1) carotenoids, particularly for salmonid and ornamental“aquarium” fishes, to enhance flesh and skin coloration, respectively;(2) binding agents, to provide stability to the pellet and reduceleaching of nutrients into the water (e.g., beef heart, starch,cellulose, pectin, gelatin, gum arabic, locust bean, agar, carageeninand other alginates); (3) preservatives, such as antimicrobials andantioxidants, to extend the shelf-life of fish diets and reduce therancidity of the fats (e.g., vitamin E, butylated hydroxyanisole,butylated hydroxytoluene, ethoxyquin, and sodium and potassium salts ofpropionic, benzoic or sorbic acids); (4) chemoattractants andflavorings, to enhance feed palatability and its intake; and, (5) otherfeedstuffs. These other feedstuffs can include such materials as fiberand ash (for use as a filler and as a source of calcium and phosphorus,respectively) and vegetable matter and/or fish or squid meal (e.g.,live, frozen or dried algae, brine shrimp, rotifers or otherzooplankton) to enhance the nutritional value of the diet and increaseits acceptance by the fish. Nutrient Requirements of Fish (NationalResearch Council, National Academy: Washington D.C., 1993) providesdetailed descriptions of the essential nutrients for fish and thenutrient content of various ingredients.

The manufacture of aquafeed formulations requires consideration of avariety of factors, since a complete diet must be nutritionallybalanced, palatable, water stable, and have the proper size and texture.With regard to nutrient composition of aquafeeds, one is referred to:Handbook on Ingredients for Aquaculture Feeds (Hertrampf, J. W. and F.Piedad-Pascual. Kluwer Academic: Dordrecht, The Netherlands, 2000) andStandard Methods for the Nutrition and Feeding of Farmed Fish and Shrimp(Tacon, A. G. J. Argent Laboratories: Redmond, 1990). In general, feedsare formulated to be dry (i.e., final moisture content of 6-10%),semi-moist (i.e., 35-40% water content) or wet (i.e., 50-70% watercontent). Dry feeds include the following: simple loose mixtures of dryingredients (i.e., “mash” or “meals”); compressed pellets, crumbles orgranules; and flakes. Depending on the feeding requirements of the fish,pellets can be made to sink or float. Semi-moist and wet feeds are madefrom single or mixed ingredients (e.g., trash fish or cooked legumes)and can be shaped into cakes or balls.

It is contemplated that the present engineered strains of Yarrowialipolytica that are producing high concentrations of ARA will beespecially useful to include in most aquaculture feeds. In addition toproviding necessary ω-6 PUFAs, the yeast itself is a useful source ofprotein that can increase the formulation's palatablility. In alternateembodiments, the oils produced by the present strains of Y. lipolyticacould be introduced directly into the aquaculture feed formulations,following extraction and purification from the cell mass.

Description of Preferred Embodiments

The present invention demonstrates the synthesis of up to 10-14% ARA inthe total lipid fraction of the oleaginous yeast, Yarrowia lipolytica.As shown in FIG. 4, numerous strains of Y. lipolytica were created byintegrating various genes into wildtype ATCC #20362 Y. lipolytica,wherein each transformant strain was capable of producing differentamounts of PUFAs (including ARA). The complete lipid profiles of strainsY2034 and Y2047 (expressing the Δ6 desaturase/Δ6 elongase pathway) andstrain Y2214 (expressing the Δ9 elongase/Δ8 desaturase pathway) areshown below in Table 10. Fatty acids are identified as 16:0, 16:1, 18:0,18:1 (oleic acid), 18:2 (LA), GLA, DGLA and ARA; and the composition ofeach is presented as a % of the total fatty acids.

TABLE 10 Lipid Profile Of Yarrowia lipolytica Strains Y2034, Y2047 AndY2214 Fatty Acid Content Strain 16:0 16:1 18:0 18:1 18:2 GLA DGLA ARAY2034 13.1 8.1 1.7 7.4 14.8 25.2 8.3 11.2 Y2047 15.9 6.6 0.7 8.9 16.629.7 0.0 10.9 Y2214 7.9 15.3 0.0 13.7 37.5 0.0 7.9 14.0

A more detailed summary of the genetic modifications contained withinstrain Y2047 is described below (wherein complete details are providedin the Examples):

-   -   (1) Expression of 1 copy of a Fusarium moniliforme Δ12        desaturase, within a FBA::F.Δ12::LIP2 chimeric gene;    -   (2) Expression of 1 copy of a synthetic Δ6 desaturase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Mortierella alpine Δ6 desaturase, within a TEF::Δ6S:LIP1        chimeric gene;    -   (3) Expression of 1 copy of a synthetic Δ5 desaturase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Homo sapiens Δ5 desaturase, within a TEF::H.D5S::PEX16 chimeric        gene;    -   (4) Expression of 1 copy of a synthetic high affinity C_(18/20)        elongase gene (codon-optimized for expression in Y. lipolytica)        derived from a Mortierella alpina high affinity C_(18/20)        elongase, within a FBAIN::EL1S::PEX20 chimeric gene;    -   (5) Expression of 1 copy of a synthetic C_(18/20) elongase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Thraustochytrium aureum C_(18/20) elongase, within a        TEF::EL2S::XPR chimeric gene; and,    -   (6) Disruption of a native Y. lipolytica Leu2 gene encoding        β-isopropylmalate dehydrogenase.

Similarly, a more detailed summary of the genetic modificationscontained within strain Y2214 is described below (wherein completedetails are provided in the Examples):

-   -   (1) Expression of 5 copies of a synthetic Δ9 elongase        (codon-optimized for expression in Y. lipolytica) derived from a        lsochrysis galbana Δ9 elongase, within GPAT:: IgD9e::PEX20,        TEF::IgD9e::LIP1, and FBAINm:: D9e::OCT chimeric genes;    -   (2) Expression of 3 copies of a synthetic Δ8 desaturase        (codon-optimized for expression in Y. lipolytica) derived from a        Euglena gracillis Δ8 desaturase, within FBAIN::D8SF::PEX16 and        GPD::D8SF::PEX16 chimeric genes;    -   (3) Expression of 2 copies of a Mortierella alpina Δ5        desaturase, within GPAT::MAΔ5::PEX20 and FBAIN::MAΔ5::PEX20        chimeric genes;    -   (4) Expression of 2 copies of a synthetic Δ5 desaturase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Isochrysis galbana Δ5 desaturase, within YAT1::I.D5S:LIP1 and        GPM/FBAIN::I.D5S::OCT chimeric genes;    -   (5) Expression of 1 copy of a Fusarium moniliforme Δ12        desaturase, within a FBAIN::F.D12S::PEX20 chimeric gene;    -   (6) Expression of 1 copy of a synthetic C_(16/18) elongase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Rattus norvegicus rELO gene, within a GPM/FBAIN::rELO2S::OCT        chimeric gene; and,    -   (7) Disruption of a native Y. lipolytica Lys5 gene encoding        saccharopine dehydrogenase.

Although the Applicants demonstrate production of 11% and 14% ARA,respectively, in these particular recombinant strains of Yarrowialipolytica, it is contemplated that the concentration of ARA in the hostcells could be dramatically increased via additional geneticmodifications, according to the invention herein. Furthermore, on thebasis of the teachings and results described herein, it is expected thatone skilled in the art will recognize the feasability and commercialutility created by using oleaginous yeast as a production platform forthe synthesis of a variety of ω-3 and/or ω-6 PUFAs, using the Δ6desaturase/Δ6 elongase pathway and/or the Δ9 elongase/Δ8 desaturasepathway.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by:

1.) Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor,N.Y. (1989) (Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W.Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofmicrobial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, 2nd ed., Sinauer Associates: Sunderland, Mass.(1989). All reagents, restriction enzymes and materials used for thegrowth and maintenance of microbial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

E. coli (XL1-Blue) competent cells were purchased from the StratageneCompany (San Diego, Calif.). E. coli strains were typically grown at 37°C. on Luria Bertani (LB) plates.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). Oligonucleotides were synthesized bySigma-Genosys (Spring, Tex.). Individual PCR amplification reactionswere carried out in a 50 μl total volume, comprising: PCR buffer(containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.75), 2 mMMgSO₄, 0.1% Triton X-100), 100 μg/mL BSA (final concentration), 200 μMeach deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μlof Pfu DNA polymerase (Stratagene, San Diego, Calif.), unless otherwisespecified. Site-directed mutagenesis was performed using Stratagene'sQuickChange™ Site-Directed Mutagenesis kit, per the manufacturers'instructions. When PCR or site-directed mutagenesis was involved insubcloning, the constructs were sequenced to confirm that no errors hadbeen introduced to the sequence. PCR products were cloned into Promega'spGEM-T-easy vector (Madison, Wis.).

DNA sequence was generated on an ABI Automatic sequencer using dyeterminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using acombination of vector and insert-specific primers. Sequence editing wasperformed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). Allsequences represent coverage at least two times in both directions.Comparisons of genetic sequences were accomplished using DNASTARsoftware (DNA Star, Inc.). Alternatively, manipulations of geneticsequences were accomplished using the suite of programs available fromthe Genetics Computer Group Inc. (Wisconsin Package Version 9.0,Genetics Computer Group (GCG), Madison, Wis.). The GCG program “Pileup”was used with the gap creation default value of 12, and the gapextension default value of 4. The GCG “Gap” or “Bestfit” programs wereused with the default gap creation penalty of 50 and the default gapextension penalty of 3. Unless otherwise stated, in all other cases GCGprogram default parameters were used.

BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J.Mol. Biol. 215:403-410 (1993) and Nucleic Acids Res. 25:3389-3402(1997)) searches were conducted to identity isolated sequences havingsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the SWISS-PROTprotein sequence database, EMBL and DDBJ databases). Sequences weretranslated in all reading frames and compared for similarity to allpublicly available protein sequences contained in the “nr” database,using the BLASTX algorithm (Gish, W. and States, D. J. Nature Genetics3:266-272 (1993)) provided by the NCBI.

The results of BLAST comparisons summarizing the sequence to which aquery sequence had the most similarity are reported according to the %identity, % similarity, and Expectation value. “% Identity” is definedas the percentage of amino acids that are identical between the twoproteins. “% Similarity” is defined as the percentage of amino acidsthat are identical or conserved between the two proteins. “Expectationvalue” estimates the statistical significance of the match, specifyingthe number of matches, with a given score, that are expected in a searchof a database of this size absolutely by chance.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “pmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s), and “kB” means kilobase(s).

Transformation and Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strains ATCC #20362, #76982 and #90812 werepurchased from the American Type Culture Collection (Rockville, Md.). Y.lipolytica strains were usually grown at 28° C. on YPD agar (1% yeastextract, 2% bactopeptone, 2% glucose, 2% agar). Alternatively, “SD”media comprises: 0.67% yeast nitrogen base with ammonium sulfate,without amino acids and 2% glucose.

Transformation of Y. lipolytica was performed according to the method ofChen, D. C. et al. (Appl. Microbiol. Biotechnol. 48(2):232-235 (1997)),unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plateand grown at 30° C. for approximately 18 hr. Several large loopfuls ofcells were scraped from the plate and resuspended in 1 mL oftransformation buffer containing: 2.25 mL of 50% PEG, average MW 3350;0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 μgsheared salmon sperm DNA. Then, approximately 500 ng of linearizedplasmid DNA was incubated in 100 μl of resuspended cells, and maintainedat 39° C. for 1 hr with vortex mixing at 15 min intervals. The cellswere plated onto selection media plates and maintained at 30° C. for 2to 3 days.

For selection of transformants, SD medium or minimal medium (“MM”) wasgenerally used; the composition of MM is as follows: 0.17% yeastnitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammoniumsulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1). Supplementsof adenine, leucine, lysine and/or uracil were added as appropriate to afinal concentration of 0.01% (thereby producing “MMA”, “MMLe”, “MMLy”and “MMU” selection media, each prepared with 20 g/L agar).

Alternatively, transformants were selected on 5-fluoroorotic acid(“FOA”; also 5-fluorouracil-6-carboxylic acid monohydrate) selectionmedia, comprising: 0.17% yeast nitrogen base (DIFCO Laboratories)without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange,Calif.) and 20 g/L agar.

Finally, for the “two-stage growth conditions” designed to promoteconditions of oleaginy, High Glucose Media (“HGM”) was prepared asfollows: 14 g/L KH₂PO₄, 4 g/LK₂HPO₄, 2 g/L MgSO₄.7H₂O, 80 g/L glucose(pH 6.5). Strains were cultured under “two-stage growth conditions”according to the following protocol: first, cells were grown intriplicate in liquid MM at 30° C. with shaking at 250 rpm/min for 48hrs. The cells were collected by centrifugation and the liquidsupernatant was extracted. The pelleted cells were resuspended in HGMand grown for either 72 hrs or 96 hrs at 30° C. with shaking at 250rpm/min. The cells were again collected by centrifugation and the liquidsupernatant was extracted.

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid analysis, cells were collected by centrifugation andlipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can.J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters wereprepared by transesterification of the lipid extract with sodiummethoxide (Roughan, G., and Nishida I. Arch Biochem Biophys.276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Example 1 Identification of Promoters for High Expression in Yarrowialipolytica

Comparative studies investigating the promoter activities of the TEF,GPD, GPDIN, GPM, GPAT, FBA, FBAIN and YAT1 promoters were performed, bysynthesizing constructs comprising each promoter and the E. coli geneencoding β-glucuronidase (GUS) as a reporter gene (Jefferson, R. A.Nature. 14(342):837-838 (1989)). Then, GUS activity was measured byhistochemical and fluorometric assays (Jefferson, R. A. Plant Mol. Biol.Reporter 5:387-405 (1987)) and/or by using Real Time PCR for mRNAquantitation.

Construction of Plasmids Comprising a Chimeric Promoter::GUS::XPR Gene

Plasmid pY5-30 (FIG. 5A; SEQ ID NO:113) contained: a Yarrowia autonomousreplication sequence (ARS18); a ColE1 plasmid origin of replication; anampicillin-resistance gene (Amp^(R)), for selection in E. coli; aYarrowia LEU2 gene, for selection in Yarrowia; and a chimericTEF::GUS::XPR gene. Based on this plasmid, a series of plasmids werecreated wherein the TEF promoter was replaced with a variety of othernative Y. lipolytica promoters.

The putative promoter regions were amplified by PCR, using the primersshown below in Table 11 and either genomic Y. lipolytica DNA as templateor a fragment of genomic DNA containing an appropriate region of DNAcloned into the pGEM-T-easy vector (Promega, Madison, Wis.).

TABLE 11 Construction of Plasmids Comprising A ChimericPromoter::GUS::XPR Gene Promoter Primers Location With Respect to GeneRE Sites Plasmid Name GPD YL211, YL212 −968 bp to the ‘ATG’ SalI andpYZGDG (SEQ ID NOs: translation initiation site NcoI 169 and 170) of thegpd gene (SEQ ID NO: 158) GPDIN YL376, YL377 −973 bp to +201 bpPstI/NcoI pDMW222 (SEQ ID NOs: around the the gpd gene (thereby (forpromoter) 171 and 172) including a 146 bp intron and PstI/SalI whereinthe intron is located (for vector) at position +49 bp to +194 bp) (SEQID NO: 159) GPM YL203, YL204 −875 bp to the ‘ATG’ NcoI and pYZGMG (SEQID NOs: translation initiation site of SalI 173 and 174) the gpm gene(SEQ ID NO: 160) GPAT GPAT-5-1, GPAT-5-2 −1130 bp to the ‘ATG’ SalI andpYGPAT-GUS (SEQ ID NOs: translation initiation site of NcoI 175 and 176)the gpat gene (SEQ ID NO: 164) FBA ODMW314, YL341 −1001 bp to −1 bparound NcoI and pDMW212 (SEQ ID NOs: the fba gene (SEQ ID NO: 161) SalI177 and 178) FBAIN ODMW320, ODMW341 −804 bp to +169 bp NcoI and pDMW214(SEQ ID NOs: around the fba gene (thereby SalI 179 and 180) including a102 bp intron wherein the intron is located at position +62 bp to +165bp) (SEQ ID NO: 162) YAT1 27203-F, 27203-R −778 bp to −1 bp aroundHindIII and pYAT-GUS (SEQ ID NOs: the yat1 gene (SEQ ID NO: 165) SalI;also 181 and 182) NcoI and HindIII Note: The ‘A’ nucleotide of the ‘ATG’translation initiation codon was designated as +1.

The individual PCR amplification reactions for GPD, GPDIN, GPM, FBA andFBAIN were carried out in a 50 μl total volume, as described in theGeneral Methods. The thermocycler conditions were set for 35 cycles at95° C. for 1 min, 56° C. for 30 sec and 72° C. for 1 min, followed by afinal extension at 72° C. for 10 min.

The PCR amplification for the GPAT promoter was carried out in a 50 μltotal volume using a 1:1 dilution of a premixed 2×PCR solution (TaKaRaBio Inc., Otsu, Shiga, 520-2193, Japan). The final composition contained25 mM TAPS (pH 9.3), 50 mM KCl, 2 mM MgCl₂, 1 mM 2-mercaptoethanol, 200μM each deoxyribonucleotide triphosphate, 10 pmole of each primer, 50 ngtemplate and 1.25 U of TaKaRa Ex Taq™ DNA polymerase (Takara Mirus Bio,Madison, Wis.). The thermocycler conditions were set for 30 cycles at94° C. for 2.5 min, 55° C. for 30 sec and 72° C. for 2.5 min, followedby a final extension at 72° C. for 6 min.

The PCR amplification for the YAT1 promoter was carried out in acomposition comparable to that described above for GPAT. The reactionmixture was first heated to 94° C. for 150 sec. Amplification wascarried out for 30 cycles at 94° C. for 30 sec, 55° C. for 30 sec and72° C. for 1 min, followed by a final extension for 7 min at 72° C.

Each PCR product was purified using a Qiagen PCR purification kit andthen digested with restriction enzymes (according to the Table aboveusing standard conditions) and the digested products were purifiedfollowing gel electrophoresis in 1% (w/v) agarose. The digested PCRproducts (with the exception of those from YAT1) were then ligated intosimilarly digested pY5-30 vector. Ligated DNA from each reaction wasthen used to individually transform E. coli Top10, E. coli DH10B or E.coli DH5a. Transformants were selected on LB agar containing ampicillin(100 μg/mL).

YAT1 required additional manipulation prior to cloning into pY5-30.Specifically, upon digestion of the YAT1 PCR product with HindIII andSalI, a ˜600 bp fragment resulted; digestion with NcoI and HindIIIresulted in a ˜200 bp fragment. Both products were isolated andpurified. Then, plasmid pYGPAT-GUS was digested with SalI and NcoI, anda ˜9.5 kB fragment was isolated and purified. The three DNA fragmentswere ligated together to create pYAT-GUS.

Analysis of the plasmid DNA from each transformation reaction confirmedthe presence of the expected plasmid. These plasmids were designated asfollows: pYZGDG (comprising a GPD::GUS::XPR chimeric gene), pDMW222(comprising a GPDIN::GUS::XPR chimeric gene), pYZGMG (comprising aGPM::GUS::XPR chimeric gene), pYGPAT-GUS (comprising a GPAT::GUS::XPRchimeric gene), pDMW212 (comprising a FBA::GUS::XPR chimeric gene),pDMW214 (comprising a FBAIN::GUS::XPR chimeric gene) and pYAT-GUS(comprising a YAT1::GUS::XPR chimeric gene).

Each of the plasmids above, and additionally plasmid pY5-30 (comprisinga TEF::GUS::XPR chimeric gene), was transformed separately into Y.lipolytica as described in the General Methods. The Y. lipolytica hostwas either Y. lipolytica ATCC #76982 or Y. lipolytica ATCC #20362,strain Y2034 (infra [Example 4], capable of producing 10% ARA via the Δ6desaturase/Δ6 elongase pathway). All transformed cells were plated ontominimal media plates lacking leucine and maintained at 30° C. for 2 to 3days.

Comparative Analysis of Yarrowia Promoters by Histochemical Analysis ofGUS Expression

Yarrowia lipolytica ATCC #76982 strains containing plasmids pY5-30,pYZGDG, pYZGMG, pDMW212 and pDMW214 were grown from single colonies in 3mL MM at 30° C. to an OD₆₀₀ ˜1.0. Then, 100 μl of cells were collectedby centrifugation, resuspended in 100 μl of histochemical stainingbuffer, and incubated at 30° C. Staining buffer was prepared bydissolving 5 mg of 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) in 50μl dimethyl formamide, followed by the addition of 5 mL 50 mM NaPO₄, pH7.0. The results of histochemical staining (FIG. 5B) showed that the TEFpromoter in construct pY5-30, the GPD promoter in construct pYZGDG, theGPM promoter in construct pYZGMG, the FBA promoter in construct pDMW212,and the FBAIN promoter in construct pDMW214 were all active. Both theFBA and FBAIN promoters appeared to be much stronger than all the otherpromoters, with the FBAIN promoter having the strongest promoteractivity.

In a separate experiment, Y. lipolytica Y2034 strains containingplasmids pY5-30, pYGPAT-GUS, pYAT-GUS and pDMW214 were grown from singlecolonies in 5 mL SD media at 30° C. for 24 hrs to an OD₆₀₀ ˜8.0. Then, 1mL of cells were collected by centrifugation. The remaining cultureswere centrifuged and washed 2× with HGM, resuspended in 5 mL each of HGMand allowed to grow at 30° C. further. After 24 and 120 hrs, ˜0.25 mL ofeach culture were centrifuged to collect the cells. Cell samples wereresuspended individually in 100 μl of histochemical staining buffer(supra). Zymolase 20T (5 μl of 1 mg/mL; ICN Biomedicals, Costa Mesa,Calif.) was added to each, and the mixture incubated at 30° C.

The results of histochemical staining showed that the GPAT promoter inconstruct pYGPAT-GUS was active, as was the YAT1 promoter in constructpYAT-GUS, when grown in SD medium for 24 hrs (FIG. 5C, “24 hr in SDmedium”). Comparatively, the GPAT promoter appeared to be much strongerthan the TEF promoter and had diminished activity with respect to theFBAIN promoter. Likewise, the YAT1 promoter appeared to be stronger thanthe TEF promoter but significantly weaker than the FBAIN promoter andGPAT promoter, when cells were grown in SD medium for 24 hrs. Moreinterestingly, however, it appeared that the YAT1 promoter was strongerthan the GPAT promoter and comparable with the FBAIN promoter in cellsgrown in HGM for 24 hrs (FIG. 5C, “24 hr in HG medium”). This remainedtrue after 120 hrs in HGM (FIG. 5C, “120 hr in HG medium”). Thus, theYAT1 promoter appeared to be induced in HGM, a medium that promotesoleaginous growth conditions due to nitrogen limitation.

Comparative Analysis of Yarrowia Promoters by Fluorometric Assay of GUSExpression

GUS activity was also assayed by fluorometric determination of theproduction of 4-methylumbelliferone (4-MU) from the correspondingsubstrate β-glucuronide (Jefferson, R. A. Plant Mol. Biol. Reporter5:387-405 (1987)).

Yarrowia lipolytica ATCC #76982 strains containing plasmids pY5-30,pYZGDG, pYZGMG, pDMW212 and pDMW214 were grown from single colonies in 3mL MM (as described above) at 30° C. to an OD₆₀₀ ˜1.0. Then, the 3 mLcultures were each added to a 500 mL flask containing 50 mL MM and grownin a shaking incubator at 30° C. for about 24 hrs. The cells werecollected by centrifugation, resuspended in Promega Cell Lysis Bufferand lysed using the BIO 101 Biopulverizer system (Vista, Calif.). Aftercentrifugation, the supernatants were removed and kept on ice.

Similarly, Y. lipolytica strain Y2034 containing plasmids pY5-30,pYAT-GUS, pYGPAT-GUS and pDMW214 constructs, respectively, were grownfrom single colonies in 10 mL SD medium at 30° C. for 48 hrs to an OD₆₀₀˜5.0. Two mL of each culture was collected for GUS activity assays, asdescribed below, while 5 mL of each culture was switched into HGM.

Specifically, cells from the 5 mL aliquot were collected bycentrifugation, washed once with 5 mL of HGM and resuspended in HGM. Thecultures in HGM were then grown in a shaking incubator at 30° C. for 24hrs. Two mL of each HGM culture were collected for the GUS activityassay, while the remaining culture was allowed to grow for an additional96 hrs before collecting an additional 2 mL of each culture for theassay.

Each 2 mL culture sample in SD medium was resuspended in 1 mL of 0.5×cell culture lysis reagent (Promega). Resuspended cells were mixed with0.6 mL of glass beads (0.5 mm diameter) in a 2.0 mL screw cap tube witha rubber O-ring. The cells were then homogenized in a Biospec minibeadbeater (Bartlesville, Okla.) at the highest setting for 90 sec. Thehomogenization mixtures were centrifuged for 2 min at 14,000 rpm in anEppendof centrifuge to remove cell debris and beads. The supernatant wasused for GUS assay and protein determination.

For each fluorometric assay, 100 μl of extract was added to 700 μl ofGUS assay buffer (2 mM 4-methylumbelliferyl-β-D-glucuronide (“MUG”) inextraction buffer) or 200 μl of extract was added to 800 μl of GUS assaybuffer. The mixtures were placed at 37° C. Aliquots of 100 μl were takenat 0, 30 and 60 min time points and added to 900 μl of stop buffer (1 MNa₂CO₃). Each time point was read using a CytoFluor Series 4000Fluorescence Multi-Well Plate Reader (PerSeptive Biosystems, Framingham,Mass.) set to an excitation wavelength of 360 nm and an emissionwavelength of 455 nm. Total protein concentration of each sample wasdetermined using 10 μl of extract and 200 μl of BioRad Bradford reagentor 20 μl of extract and 980 μl of BioRad Bradford reagent (Bradford, M.M. Anal. Biochem. 72:248-254 (1976)). GUS activity was expressed asnmoles of 4-MU per minute per mg of protein.

Results of these fluorometric assays designed to compare the TEF, GPD,GPM, FBA and FBAIN promoters in Y. lipolytica ATCC #76982 strains areshown in FIG. 6A. Specifically, the FBA promoter was 2.2 times strongerthan the GPD promoter in Y. lipolytica. Additionally, the GUS activityof the FBAIN promoter was about 6.6 times stronger than the GPDpromoter.

Results of these fluorometric assays designed to compare the TEF, GPAT,YAT1 and FBAIN promoters in Y. lipolytica strain Y2034 are shown in theTable below.

TABLE 12 Comparison of TEF, FBAIN, YAT1 And GPAT Promoter- ActivityUnder Various Growth Conditions Culture Promoter Conditions TEF FBAINYAT1 GPAT  48 hr, SD 0.401 43.333 0.536 5.252  24 hr, HGM 0.942 30.69419.154 2.969 120 hr HGM 0.466 17.200 13.400 3.050

Based on the data above wherein the activity of the YAT1 promoter wasquantitated based on GUS activity of cell extracts, the activity of theYAT1 promoter increased by ˜37 fold when cells were switched from SDmedium into HGM and grown for 24 hrs. After 120 hrs in HGM, the activitywas reduced somewhat but was still 25× higher than the activity in SDmedium. In contrast, the activity of the FBAIN promoter and the GPATpromoter was reduced by 30% and 40%, respectively, when switched from SDmedium into HGM for 24 hrs. The activity of the TEF promoter increasedby 2.3 fold after 24 hrs in HGM. Thus, the YAT1 promoter is inducibleunder oleaginous conditions.

Comparative Analysis of Yarrowia Promoters by Quantitative PCR Analysesof GUS Expression

The transcriptional activities of the TEF, GPD, GPDIN, FBA and FBAINpromoters were determined in Y. lipolytica containing the pY5-30,pYZGDG, pDMW222, pDMW212 and pDMW214 constructs by quantitative PCRanalyses. This required isolation of RNA and real time RT-PCR.

More specifically, Y. lipolytica ATCC #76982 strains containing pY5-30,pYZGDG, pDMW222, pDMW212 and pDMW214 were grown from single colonies in6 mL of MM in 25 mL Erlenmeyer flasks for 16 hrs at 30° C. Each of the 6mL starter cultures was then added to individual 500 mL flaskscontaining 140 mL HGM and incubated at 30° C. for 4 days. In eachinterval of 24 hrs, 1 mL of each culture was removed from each flask tomeasure the optical density, 27 mL was removed and used for afluorometric GUS assay (as described above), and two aliquots of 1.5 mLwere removed for RNA isolation. The culture for RNA isolation wascentrifuged to produce a cell pellet.

The RNA was isolated from Yarrowia strains according to the modifiedQiagen RNeasy mini protocol (Qiagen, San Diego, Calif.). Briefly, ateach time point for each sample, 340 μL of Qiagen's buffer RLT was usedto resuspend each of the two cell pellets. The buffer RLT/cellsuspension mixture from each of the two tubes was combined in a beadbeating tube (Bio101, San Diego, Calif.). About 500 μL of 0.5 mL glassbeads was added to the tube and the cells were disrupted by bead beating2 min at setting 5 (BioPulverizer, Bio101 Company, San Diego, Calif.).The disrupted cells were then pelleted by centrifugation at 14,000 rpmfor 1 min and 350 μl of the supernatant was transferred to a newmicrocentrifuge tube. Ethanol (350 μL of 70%) was added to eachhomogenized lysate. After gentle mixing, the entire sample was added toa RNeasy mini column in a 2 mL collection tube. The sample wascentrifuged for 15 sec at 10,000 rpm. Buffer RW1 (350 μL) was added tothe RNeasy mini column and the column was centrifuged for 15 sec at10,000 rpm to wash the cells. The eluate was discarded. Qiagen's DNase1stock solution (10 μL) was added to 70 μl of Buffer RDD and gentlymixed. This entire DNase solution was added to the RNeasy mini columnand incubated at room temperature for 15 min. After the incubation step,350 μL of Buffer RW1 was added to the mini column and the column wascentrifuged for 15 sec at 10,000 rpm. The column was washed twice with700 μL Buffer RW1. RNase-free water (50 μL) was added to the column. Thecolumn was centrifuged for 1 min at 10,000 rpm to elute the RNA.

A two-step RT-PCR protocol was used, wherein total Yarrowia RNA wasfirst converted to cDNA and then cDNA was analyzed using Real Time PCR.The conversion to cDNA was performed using Applied Biosystems' HighCapacity cDNA Archive Kit (PN#4322171; Foster City, Calif.) andMolecular Biology Grade water from MediaTech, Inc. (PN#46-000-Con; HollyHill, Fla.). Total RNA from Yarrowia (100 ng) was converted to cDNA bycombining it with 10 μl of RT buffer, 4 μl of 25×dNTPs, 10 μl 10× RandomHexamer primers, 5 μl Multiscribe Reverse Transcriptase and 0.005 μlRNase Inhibitor, and brought to a total reaction volume of 100 μl withwater. The reactions were incubated in a thermocycler for 10 min at 25°C. followed by 2 hrs at 37° C. The cDNA was stored at −20° C. prior toReal Time analysis.

Real Time analysis was performed using the SYBR Green PCR Master Mixfrom Applied Biosystems (PN#4309155). The Reverse Transcription reaction(2 μl) was added to 10 μl of 2×SYBR PCR Mix, 0.2 μl of 100 μM Forwardand Reverse primers for either URA (i.e., primers YL-URA-16F andYL-URA-78R [SEQ ID NOs:183 and 184]) or GUS (i.e., primers GUS-767F andGUS-891R [SEQ ID NO:185 and 186]) and 7.2 μl water. The reactions werethermocycled for 10 min at 95° C. followed by 40 cycles of 95° C. for 5sec and 60° C. for 1 min in an ABI 7900 Sequence Detection Systeminstrument. Real time fluorescence data was collected during the 60° C.extension during each cycle.

Relative quantitation was performed using the ΔΔCT method as per UserBulletin #2: “Relative Quantitation of Gene Expression”, AppliedBiosystems, Updated October 2001. The URA gene was used fornormalization of GUS expression. In order to validate the use of URA asa normalizer gene, the PCR efficiency of GUS and URA were compared andthey were found to be 1.04 and 0.99, respectively (where 1.00 equals100% efficiency). Since the PCR efficiencies were both near 100%, theuse of URA as a normalizer for GUS expression was validated, as was theuse of the ΔΔCT method for expression quantitation. The normalizedquantity is referred to as the ΔCT.

The GUS mRNA in each different strain (i.e., Y. lipolytica ATCC #76982strains containing the pYZGDG, pDMW222, pDMW212 and pDMW214 constructs)was quantified to the mRNA level of the strain with pY5-30 (TEF::GUS).Thus, relative quantitation of expression was calculated using the mRNAlevel of the strain with TEF::GUS as the reference sample. Thenormalized value for GPD::GUS, GPDIN::GUS, FBA::GUS and FBAIN::GUS wascompared to the normalized value of the TEF::GUS reference. Thisquantity is referred to as the ΔΔCT. The ΔΔCT values were then convertedto absolute values by utilizing the formula 2^(−ΔΔCT). These valuesrefer to the fold increase in the mRNA level of GUS in the strainscomprising the chimeric GPD::GUS, GPDIN::GUS, FBA::GUS and FBAIN::GUSgenes, as compared to the chimeric TEF::GUS gene. Using thismethodology, it was possible to compare the activity of the TEF promoterto the GPD, GPDIN, FBA and FBAIN promoters.

The results of the relative quantitation of mRNA for each GUS chimericgene are shown in FIG. 6B. More specifically, the assay showed thatafter 24 hrs in HGM, the transcription activity of FBA and FBAINpromoters was about 3.3 and 6 times stronger than the TEF promoter,respectively. Similarly, the transcription activity of the GPD and GPDINpromoters is about 2 and 4.4 times stronger than the TEF promoter,respectively. While the transcription activities of the FBA::GUS,FBAIN::GUS, GPD::GUS and GPDIN::GUS gene fusion decreased over the 4 dayperiod of the experiment, the transcriptional activity of the FBAIN andGPDIN promoters was still about 3 and 2.6 times stronger than the TEFpromoter in the final day of the experiment.

Example 2 Identification of Enhancers Useful to Increase GeneTranscription in Yarrowia lipolytica

Based on the strong promoter activities of FBAIN and GPDIN (whereinactivity was greater than that of the FBA and GPD promoters,respectively) and the identification of an intron within each promoterregion, the present work was conducted to determine whether enhancerswere present in each intron.

Specifically, two chimeric promoters consisting of a GPM::FBAIN promoterfusion and a GPM::GPDIN promoter fusion were generated to driveexpression of the GUS reporter gene. The chimeric promoters (comprisedof a “component 1” and a “component 2”) are described below in Table 13.

TABLE 13 Construction of Plasmids Comprising A Chimeric Promoter WithinA Chimeric Promoter::GUS::XPR Gene Chimeric Plasmid Promoter Component 1Component 2 Name GPM::FBAIN −1 bp to +1 bp to +171 bp pDMW224 (SEQ ID−843 bp region of FBAIN, NO: 167) region wherein the intron is of GPMlocated at position +62 bp to +165 bp GPM::GPDIN −1 bp to +1 bp to +198bp pDMW225 (SEQ ID −843 bp region of GPDIN, NO: 168) region wherein theintron is of GPM located at position +49 bp to +194 bpThe chimeric promoters were positioned such that each drove expressionof the GUS reporter gene in plasmids pDMW224 and pDMW225.

The activities of the GPM::FBAIN promoter and the GPM::GPDIN promoterwere compared with the TEF, FBAIN, GPDIN and GPM promoters by comparingthe GUS activity in Y. lipolytica strains comprising pDMW224 and pDMW225relative to the GUS activity in Y. lipolytica strains comprising pY5-30,pYZGDG, pYZGMG and pDMW214 constructs based on results fromhistochemical assays (as described in Example 1). As previouslydetermined, the FBAIN promoter was the strongest promoter. However, thechimeric GPM::FBAIN promoter and the chimeric GPM::GPDIN promoter wereboth much stronger than the GPM promoter and appeared to be equivalentin activity to the GPDIN promoter. Thus, this confirmed the existence ofan enhancer in both the GPDIN promoter and the FBAIN promoter.

One skilled in the art would readily be able to construct similarchimeric promoters, using either the GPDIN intron or the FBAIN intron.

Example 3 Sulfonylurea Selection

Genetic improvement of Yarrowia has been hampered by the lack ofsuitable non-antibiotic selectable transformation markers. The presentExample describes the development of a dominant, non antibiotic markerfor Y. lipolytica based on sulfonylurea resistance that is alsogenerally applicable to industrial yeast strains that may be haploid,diploid, aneuploid or heterozygous.

Theory and Initial Sensitivity Screening

Acetohydroxyacid synthase (AHAS) is the first common enzyme in thepathway for the biosynthesis of branched-chain amino acids. It is thetarget of the sulfonylurea and imidazolinone herbicides. As such,sulfonyl urea herbicide resistance has been reported in both microbesand plants. For example, in Saccharomyces cerevisiae, the single W586Lmutation in AHAS confers resistance to sulfonylurea herbicides (Falco,S. C., et al., Dev. Ind. Microbiol. 30:187-194 (1989); Duggleby, R. G.,et. al. Eur. J. Biochem. 270:2895 (2003)).

When the amino acid sequences of wild type AHAS Y. lipolytica (GenBankAccession No. XP_(—)501277) and S. cerevisiae (GenBank Accession No.P07342) enzymes were aligned, the Trp amino acid residue at position 586of the S. cerevisiae enzyme was equivalent to the Trp residue atposition 497 of the Y. lipolytica enzyme. It was therefore hypothesizedthat W497L mutation in the Y. lipolytica enzyme would likely confersulfonylurea herbicide resistance, if the wild type cells werethemselves sensitive to sulfonylurea. Using methodology well known tothose of skill in the art, it was determined that sulfonylurea(chlorimuron ethyl) at a concentration of 100 μg/mL in minimal mediumwas sufficient to inhibit growth of wild type Y. lipolytica strains ATCC#20362 and ATCC #90812.

Synthesis of a Mutant W497L AHAS Gene

The Y. lipolytica AHAS gene containing the W497L mutation (SEQ IDNO:280) was created from genomic DNA in a two-step reaction. First, the5′ portion of the AHAS gene was amplified from genomic DNA using PfuUltra™ High-Fidelity DNA Polymerase (Stratagene, Catalog #600380) andprimers 410 and 411 [SEQ ID NOs:365 and 366]; the 3′ portion of the genewas amplified similarly using primers 412 and 413 [SEQ ID NOs:367 and368]. The two pairs of primers were overlapping such that theoverlapping region contained the W497L mutation (wherein the mutationwas a ‘CT’ change to ‘TG’).

The 5′ and 3′ PCR products of the correct size were gel purified andused as the template for the second round of PCR, wherein the entiremutant gene was amplified using primers 414 and 415 (SEQ ID NOs:369 and370) and a mixture of the products from the two primary PCR reactions.This mutant gene carried its own native promoter and terminatorsequences. The second round PCR product of the correct size was gelpurified and cloned by an in-fusion technique into the vector backboneof plasmid pY35 [containing a chimeric TEF::Fusarium moniliforme Δ12desaturase (Fm2) gene, the E. coli origin of replication, a bacterialampicillin resistance gene, the Yarrowia Leu 2 gene and the Yarrowiaautonomous replication sequence (ARS); see WO 2005/047485 for additionaldetails], following its digestion with enzymes SalI/BsiWI. The in-fusionreaction mixture was transformed into TOP10 competent cells (Invitrogen,Catalog #C4040-10). After one day selection on LB/Amp plates, eight (8)colonies were analyzed by DNA miniprep. Seven clones were confirmed tobe correct by restriction digest. One of them that contained thesulfonylurea resistance gene as well as the LEU gene was designated“pY57” (or “pY57.YI.AHAS.w4971”; FIG. 7A).

Wild type Y. lipolytica strains ATCC #90812 and #20362 were transformedwith pY57 and ‘empty’ LEU by a standard Lithium Acetate method.Transformation controls comprising ‘No-DNA’ were also utilized.Transformants were plated onto either MM or MM+sulfonylurea (SU; 100μg/mL) agar plates and the presence or absence of colonies was evaluatedfollowing four days of growth.

TABLE 14 AHAS Selection In Yarrowia lipolytica ATCC #90812 ATCC #20362MM + SU MM + SU Plasmid MM (100 μg/mL) MM (100 μg/mL) pY57 coloniescolonies colonies colonies Leu vector colonies No colonies colonies Nocolonies control No DNA No colonies No colonies No colonies No coloniescontrol

Based on the results shown above, AHAS W497L was a good non-antibioticselection marker in both Y. lipolytica ATCC #90812 and #20362.Subsequently, Applicants used a sulfonylurea concentration of 150 μg/mL.This new marker is advantageous for transforming Y. lipolytica since itdoes not rely on a foreign gene but on a mutant native gene and itneither requires auxotrophy nor results in auxotrophy. The herbicide isnon-toxic to humans and animals.

It is expected that this selection method will be generally applicableto other industrial yeast strains that may be haploid, diploid,aneuploid or heterozygous, if mutant AHAS enzymes were created in amanner analogous to that described herein.

Example 4 Δ6 Desaturase/Δ6 Elongase Pathway: Generation of Y2034 andY2047 Strains to Produce about 10-11% ARA of Total Lipids

The present Example describes the construction of strains Y2034 andY2047, derived from Yarrowia lipolytica ATCC #20362, capable ofproducing 10 and 11% ARA, respectively, relative to the total lipids(FIG. 4). These strains were both engineered to express the Δ6desaturase/Δ6 elongase pathway; thus, it was not unexpected thatanalysis of the complete lipid profiles of strains Y2034 and Y2047indicated co-synthesis of ˜25-29% GLA.

The development of strains Y2034 and Y2047 first required theconstruction of strain M4 (producing 8% DGLA).

Generation of M4 Strain to Produce about 8% DGLA of Total Lipids

Construct pKUNF12T6E (FIG. 7B; SEQ ID NO:114) was generated to integratefour chimeric genes (comprising a Δ12 desaturase, a Δ6 desaturase andtwo C_(18/20) elongases) into the Ura3 loci of wild type Yarrowia strainATCC #20362, to thereby enable production of DGLA. The pKUNF12T6Eplasmid contained the following components:

TABLE 15 Description of Plasmid pKUNF12T6E (SEQ ID NO: 114) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:114 Components AscI/BsiWI 784 bp 5′ part of Yarrowia Ura3 gene (GenBank(9420-8629) Accession No. AJ306421) SphI/PacI 516 bp 3′ part of YarrowiaUra3 gene (GenBank (12128-1) Accession No. AJ306421) SwaI/BsiWIFBAIN::EL1S::Pex20, comprising: (6380-8629) FBAIN: FBAIN promoter (SEQID NO: 162) EL1S: codon-optimized elongase 1 gene (SEQ ID NO: 19),derived from Mortierella alpina (GenBank Accession No. AX464731) Pex20:Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank AccessionNo. AF054613) BglII/SwaI TEF:: Δ6S::Lip1, comprising: (4221-6380) TEF:TEF promoter (GenBank Accession No. AF054508) Δ6S: codon-optimized Δ6desaturase gene (SEQ ID NO: 3), derived from Mortierella alpina (GenBankAccession No. AF465281) Lip1: Lip1 terminator sequence from YarrowiaLip1 gene (GenBank Accession No. Z50020) PmeI/ClaI FBA::F.Δ12::Lip2,comprising: (4207-1459) FBA: FBA promoter (SEQ ID NO: 161) F.Δ12:Fusarium moniliforme Δ12 desaturase gene (SEQ ID NO: 27) Lip2: Lip2terminator sequence from Yarrowia Lip2 gene (GenBank Accession No.AJ012632) ClaI/PacI TEF::EL2S::XPR, comprising: (1459-1) TEF: TEFpromoter (GenBank Accession No. AF054508) EL2S: codon-optimized elongasegene (SEQ ID NO: 22), derived from Thraustochytrium aureum (U.S. Pat.No. 6,677,145) XPR: ~100 bp of the 3′ region of the Yarrowia Xpr gene(GenBank Accession No. M17741)

The pKUNF12T6E plasmid was digested with AscI/SphI, and then used fortransformation of wild type Y. lipolytica ATCC #20362 according to theGeneral Methods. The transformant cells were plated onto FOA selectionmedia plates and maintained at 30° C. for 2 to 3 days. The FOA resistantcolonies were picked and streaked onto MM and MMU selection plates. Thecolonies that could grow on MMU plates but not on MM plates wereselected as Ura− strains. Single colonies of Ura− strains were theninoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2days.

The cells were collected by centrifugation, lipids were extracted, andfatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed the presence of DGLA in the transformants containingthe 4 chimeric genes of pKUNF12T6E, but not in the wild type Yarrowiacontrol strain. Most of the selected 32 Ura⁻ strains produced about 6%DGLA of total lipids. There were 2 strains (i.e., strains M4 and 13-8)that produced about 8% DGLA of total lipids.

Generation of Y2034 and Y2047 Strains to Produce about 10% ARA of TotalLipids

Constructs pDMW232 (FIG. 7C; SEQ ID NO:115) and pDMW271 (FIG. 7D; SEQ IDNO:116) were generated to integrate either two or three Δ5 chimericgenes into the Leu2 gene of Yarrowia strain M4, respectively.

The plasmids pDMW232 and pDMW271 contained the following components, asdescribed in Tables 16 and 17, respectively:

TABLE 16 Description of Plasmid pDMW232 (SEQ ID NO: 115) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:115 Components AscI/BsiWI 788 bp 5′ part of Yarrowia Leu2 gene (GenBank(5550-4755) Accession No. AF260230) SphI/PacI 703 bp 3′ part of YarrowiaLeu2 gene (GenBank (8258-8967) Accession No. AF260230) SwaI/BsiWIFBAIN::MAΔ5::Pex20, comprising: (2114-4755) FBAIN: FBAIN promoter (SEQID NO: 162) MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ ID NO: 6)(GenBank Accession No. AF067654) Pex20: Pex20 terminator sequence ofYarrowia Pex20 gene (GenBank Accession No. AF054613) SwaI/ClaITEF::MAΔ5::Lip1, comprising: (2114-17) TEF: TEF promoter (GenBankAccession No. AF054508) MAΔ5: SEQ ID NO: 6 (supra) Lip1: Lip1 terminatorsequence of Yarrowia Lip1 gene (GenBank Accession No. Z50020) PmeI/ClaIYarrowia Ura3 gene (GenBank Accession No. (5550-4755) AJ306421)

TABLE 17 Description of Plasmid pDMW271 (SEQ ID NO: 116) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:116 Components AscI/BsiWI 788 bp 5′ part of Yarrowia Leu2 gene (GenBank(5520-6315) Accession No. AF260230) SphI/PacI 703 bp 3′ part of YarrowiaLeu2 gene (GenBank (2820-2109) Accession No. AF260230) SwaI/BsiWIFBAIN::MAΔ5::Pex20: as described for pDMW232 (8960-6315) (supra)SwaI/ClaI TEF::MAΔ5::Lip1: as described for pDMW232 (8960-11055) (supra)PmeI/ClaI Yarrowia Ura3 gene (GenBank Accession No. (12690-11055)AJ306421) ClaI/PacI TEF::HΔ5S::Pex16, comprising: (1-2109) TEF: TEFpromoter (GenBank Accession No. AF054508) HΔ5S: codon-optimized Δ5desaturase gene (SEQ ID NO: 13), derived from Homo sapiens (GenBankAccession No. NP_037534) Pex16: Pex16 terminator sequence of YarrowiaPex16 gene (GenBank Accession No. U75433)

Plasmids pDMW232 and pDMW271 were each digested with AscI/SphI, and thenused to transform strain M4 separately according to the General Methods.Following transformation, the cells were plated onto MMLe plates andmaintained at 30° C. for 2 to 3 days. The individual colonies grown onMMLe plates from each transformation were picked and streaked onto MMand MMLe plates. Those colonies that could grow on MMLe plates but noton MM plates were selected as Leu2⁻ strains. Single colonies of Leu2⁻strains were then inoculated into liquid MMLe media at 30° C. and shakenat 250 rpm/min for 2 days. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of ARA in pDMW232 and pDMW271transformants, but not in the parental M4 strain. Specifically, amongthe 48 selected Leu2⁻ transformants with pDMW232, there were 34 strainsthat produced less than 5% ARA, 11 strains that produced 6-8% ARA, and 3strains that produced about 10% ARA of total lipids in the engineeredYarrowia. One of the strains that produced 10% ARA was named “Y2034”.

Meanwhile, of the 48 selected Leu2 transformants with pDMW271, therewere 35 strains that produced less than 5% ARA of total lipids, 12strains that produced 6-8% ARA, and 1 strain that produced about 11% ARAof total lipids in the engineered Yarrowia. The strain that produced 11%ARA was named “Y2047”.

Example 5 Generation of Intermediate Strain Y2031, Having a Ura−Genotype and Producing 45% LA of Total Lipids

Strain Y2031 was generated by integration of the TEF::Y.Δ12::Pex20chimeric gene of plasmid pKUNT2 (FIG. 8A) into the Ura3 gene locus ofwild type Yarrowia strain ATCC #20362, to thereby to generate a Ura−genotype.

Specifically, plasmid pKUNT2 contained the following components:

TABLE 18 Description of Plasmid pKUNT2 (SEQ ID NO: 117) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:117 Components AscI/BsiWI 784 bp 5′ part of Yarrowia Ura3 gene (GenBank(3225-3015) Accession No. AJ306421) SphI/PacI 516 bp 3′ part of YarrowiaUra3 gene (GenBank (5933-13) Accession No. AJ306421) EcoRI/BsiWITEF::Y.Δ12::Pex20, comprising: (6380-8629) TEF: TEF promoter (GenBankAccession No. AF054508) Y.Δ12: Yarrowia Δ12 desaturase gene (SEQ ID NO:23) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBankAccession No. AF054613)

The pKUNT2 plasmid was digested with AscI/SphI, and then used fortransformation of wild type Y. lipolytica ATCC #20362 according to theGeneral Methods. The transformant cells were plated onto FOA selectionmedia plates and maintained at 30° C. for 2 to 3 days. The FOA resistantcolonies were picked and streaked onto MM and MMU selection plates. Thecolonies that could grow on MMU plates but not on MM plates wereselected as Ura− strains. Single colonies (5) of Ura− strains were theninoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed that there were about 45% LA in two Ura− strains(i.e., strains #2 and #3), compared to about 20% LA in the wild typeATCC #20362. Transformant strain #2 was designated as strain “Y2031”.

Example 6 Synthesis and Functional Expression of a Codon-Optimized Δ9Elongase Gene in Yarrowia lipolytica

The codon usage of the Δ9 elongase gene of lsochrysis galbana (GenBankAccession No. AF390174) was optimized for expression in Y. lipolytica,in a manner similar to that described in WO 2004/101753. Specifically,according to the Yarrowia codon usage pattern, the consensus sequencearound the ATG translation initiation codon, and the general rules ofRNA stability (Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23(2001)), a codon-optimized Δ9 elongase gene was designed (SEQ ID NO:41),based on the DNA sequence of the I. galbana gene (SEQ ID NO:39). Inaddition to modification of the translation initiation site, 126 bp ofthe 792 bp coding region were modified, and 123 codons were optimized.None of the modifications in the codon-optimized gene changed the aminoacid sequence of the encoded protein (GenBank Accession No. AF390174;SEQ ID NO:40).

In Vitro Synthesis of a Codon-Optimized Δ9 Elongase Gene for Yarrowia

The codon-optimized Δ9 elongase gene was synthesized as follows. First,eight pairs of oligonucleotides were designed to extend the entirelength of the codon-optimized coding region of the I. galbana Δ9elongase gene (e.g., IL3-1A, IL3-1B, IL3-2A, IL3-2B, IL3-3A, IL3-3B,IL3-4A, IL3-4B, IL3-5A, IL3-5B, IL3-6A, IL3-6B, IL3-7A, IL3-7B, IL3-8Aand IL3-8B, corresponding to SEQ ID NOs:187-202). Each pair of sense (A)and anti-sense (B) oligonucleotides were complementary, with theexception of a 4 bp overhang at each 5′-end. Additionally, primersIL3-1F, IL3-4R, IL3-5F and IL3-8R (SEQ ID NOs:203-206) also introducedNcoI, PstI, PstI and Not1 restriction sites, respectively, forsubsequent subcloning.

Each oligonucleotide (100 ng) was phosphorylated at 37° C. for 1 hr in avolume of 20 μl containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mMDTT, 0.5 mM spermidine, 0.5 mM ATP and 10 U of T4 polynucleotide kinase.Each pair of sense and antisense oligonucleotides was mixed and annealedin a thermocycler using the following parameters: 95° C. (2 min), 85° C.(2 min), 65° C. (15 min), 37° C. (15 min), 24° C. (15 min) and 4° C. (15min). Thus, IL3-1A (SEQ ID NO:187) was annealed to IL3-1B (SEQ IDNO:188) to produce the double-stranded product “IL3-1AB”. Similarly,IL3-2A (SEQ ID NO:189) was annealed to IL3-2B (SEQ ID NO:190) to producethe double-stranded product “IL3-2AB”, etc.

Two separate pools of annealed, double-stranded oligonucleotides werethen ligated together, as shown below: Pool 1 (comprising IL3-1AB,IL3-2AB, IL3-3AB and IL3-4AB); and, Pool 2 (comprising IL3-SAB, IL3-6AB,IL3-7AB and IL3-8AB). Each pool of annealed oligonucleotides was mixedin a volume of 20 μl with 10 U of T4 DNA ligase and the ligationreaction was incubated overnight at 16° C.

The product of each ligation reaction was then used as template toamplify the designed DNA fragment by PCR. Specifically, using theligated “Pool 1” mixture (i.e., IL3-1AB, IL3-2AB, IL3-3AB and IL3-4AB)as template, and oligonucleotides IL3-1F and IL3-4R (SEQ ID NOs:203 and204) as primers, the first portion of the codon-optimized A elongasegene was amplified by PCR. The PCR amplification was carried out in a 50μl total volume, as described in the General Methods. Amplification wascarried out as follows: initial denaturation at 95° C. for 3 min,followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30sec, 72° C. for 40 sec. A final extension cycle of 72° C. for 10 min wascarried out, followed by reaction termination at 4° C. The 417 bp PCRfragment was subcloned into the pGEM-T easy vector (Promega) to generatepT9(1-4).

Using the ligated “Pool 2” mixture (i.e., IL3-SAB, IL3-6AB, IL3-7AB andIL3-8AB) as the template, and oligonucleotides IL3-5F and IL3-8R (SEQ IDNOs:205 and 206) as primers, the second portion of the codon-optimizedΔ9 elongase gene was amplified similarly by PCR and cloned intopGEM-T-easy vector to generate pT9(5-8).

E. coli was transformed separately with pT9(1-4) and pT9(5-8) and theplasmid DNA was isolated from ampicillin-resistant transformants.Plasmid DNA was purified and digested with the appropriate restrictionendonucleases to liberate the 417 bp NcoI/PstI fragment of pT9(1-4) (SEQID NO:207) and the 377 bp PstI/Not1 fragment of pT9(5-8) (SEQ IDNO:208). These two fragments were then combined and directionallyligated together with Nco1/Not1 digested pZUF17 (SEQ ID NO:118; FIG. 8B)to generate pDMW237 (FIG. 8C; SEQ ID NO:119). The DNA sequence of theresulting synthetic Δ9 elongase gene (“IgD9e”) in pDMW237 was exactlythe same as the originally designed codon-optimized gene (i.e., SEQ IDNO:41) for Yarrowia.

Expression of the Codon-Optimized Δ9 Elongase Gene in Y. lipolytica

Construct pDMW237 (FIG. 8C), an auto-replication plasmid comprising achimeric FBAIN::Ig D9e::Pex20 gene, was transformed into Y. lipolyticaY2031 strain (Example 4) as described in the General Methods. Threetransformants of Y2031 with pDMW237 were grown individually in MM mediafor two days and the cells were collected by centrifugation, lipids wereextracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

The GC results showed that there were about 7.1%, 7.3% and 7.4% EDA,respectively, produced in these transformants with pDMW237. These datademonstrated that the synthetic, codon-optimized IgD9e could convertC18:2 to EDA. The “percent (%) substrate conversion” of thecodon-optimized gene was determined to be about 13%.

Example 7 Synthesis of a Codon-Optimized Δ8 Desaturase Gene in Yarrowialipolytica

The codon usage of the Δ8 desaturase gene of Euglena gracilis (Gen BankAccession No. AAD45877) was optimized for expression in Y. lipolytica,in a manner similar to that described in WO 2004/101753 and Example 6(supra). Despite synthesis of three different codon-optimized genes(i.e., “D8s⁻¹”, “D85-2” and “D85-3”), none of the genes were capable ofdesaturating EDA to DGLA. It was therefore hypothesized that thepreviously published Δ8 desaturase sequences were incorrect and it wasnecessary to isolate the Δ8 desaturase from Euglena gracilis directly,following mRNA isolation, cDNA synthesis and PCR. This resulted in twosimilar sequences, identified herein as Eg5 (SEQ ID NOs:44 and 45) andEg12 (SEQ ID NOs:46 and 47).

Functional analysis of each gene sequence was performed by cloning thegenes into a Saccharomyces cerevisiae yeast expression vector andconducting substrate feeding trials. Although both Eg5 and Eg12 wereable to desaturase EDA and ETrA to produce DGLA and ETA, respectively,Eg5 had significantly greater activity than Eg12.

Based on the confirmed Δ8 desaturase activity of Eg5, the sequence wascodon-optimized for expression in Yarrowia lipolytica to thereby resultin the synthesis of a synthetic, functional codon-optimized Δ8desaturase designated as “D8SF” (SEQ ID NOs:48 and 49).

Preliminary In Vitro Synthesis of a Codon-Optimized Δ8 Desaturase Gene

A codon-optimized Δ8 desaturase gene (designated “D8s⁻¹”; SEQ ID NO:209)was designed, based on the published sequence of Euglena gracilis (SEQID NOs:42 and 43), according to the Yarrowia codon usage pattern (WO2004/101753), the consensus sequence around the ‘ATG’ translationinitiation codon, and the general rules of RNA stability (Guhaniyogi, G.and J. Brewer, Gene 265(1-2):11-23 (2001)). In addition to modificationof the translation initiation site, 200 bp of the 1260 bp coding regionwere modified (15.9%). None of the modifications in the codon-optimizedgene changed the amino acid sequence of the encoded protein (SEQ IDNO:43) except the second amino acid from ‘K’ to ‘E’ to add a NcoI sitearound the translation initiation codon.

Specifically, the codon-optimized Δ8 desaturase gene was synthesized asfollows. First, thirteen pairs of oligonucleotides were designed toextend the entire length of the codon-optimized coding region of the E.gracilis Δ8 desaturase gene (e.g., D8-1A, D8-1B, D8-2A, D8-2B, D8-3A,D8-3B, D8-4A, D8-4B, D8-5A, D8-5B, D8-6A, D8-6B, D8-7A, D8-7B, D8-8A,D8-8B, D8-9A, D8-9B, D8-10A, D8-10B, D8-11A, D8-11B, D8-12A, D8-12B,D8-13A and D8-13B, corresponding to SEQ ID NOs:210-235). Each pair ofsense (A) and anti-sense (B) oligonucleotides were complementary, withthe exception of a 4 bp overhang at each 5′-end. Additionally, primersD8-1A, D8-3B, D8-7A, D8-9B and D8-13B (SEQ ID NOs:210, 215, 222, 227 and235) also introduced NcoI, BglII, Xho1, SacI and Not1 restriction sites,respectively, for subsequent subcloning.

Oligonucleotides (100 ng of each) were phosphorylated as described inExample 6, and then each pair of sense and antisense oligonucleotideswas mixed and annealed together [e.g., D8-1A (SEQ ID NO:210) wasannealed to D8-1B (SEQ ID NO:211) to produce the double-stranded product“D8-1AB” and D8-2A (SEQ ID NO:212) was annealed to D8-2B (SEQ ID NO:213)to produce the double-stranded product “D8-2AB”, etc.].

Four separate pools of annealed, double-stranded oligonucleotides werethen ligated together, as shown below: Pool 1 (comprising D8-1AB, D8-2ABand D8-3AB); Pool 2 (comprising D8-4AB, D8-5AB and D8-6AB); Pool 3(comprising D8-7AB, D8-8AB, and D8-9AB); and, Pool 4 (comprisingD8-10AB, D8-11AB, D8-12AB and D8-13AB). Each pool of annealedoligonucleotides was mixed in a volume of 20 μl with 10 U of T4 DNAligase and the ligation reaction was incubated overnight at 16° C.

The product of each ligation reaction was then used as template toamplify the designed DNA fragment by PCR. Specifically, using theligated “Pool 1” mixture (i.e., D8-1AB, D8-2AB and D8-3AB) as template,and oligonucleotides D8-1F and D8-3R (SEQ ID NOs:236 and 237) asprimers, the first portion of the codon-optimized Δ8 desaturase gene wasamplified by PCR. The PCR amplification was carried out in a 50 μl totalvolume, as described in Example 6. The 309 bp PCR fragment was subclonedinto the pGEM-T easy vector (Promega) to generate pT8(1-3).

Using the ligated “Pool 2” mixture (i.e., D8-4AB, D8-5AB and D8-6AB) asthe template, and oligonucleotides D8-4F and D8-6R (SEQ ID NOs:238 and239) as primers, the second portion of the codon-optimized Δ8 desaturasegene was amplified similarly by PCR and cloned into pGEM-T-easy vectorto generate pT8(4-6). Using the ligated “Pool 3” mixture (i.e., D8-7AB,D8-8AB and D8-9AB) as the template and oligonucleotides D8-7F and D8-9R(SEQ ID NOs:240 and 241) as primers, the third portion of thecodon-optimized Δ8 desaturase gene was amplified similarly by PCR andcloned into pGEM-T-easy vector to generate pT8(7-9). Finally, using the“Pool 4” ligation mixture (i.e., D8-10AB, D8-11AB, D8-12AB and D8-13AB)as template, and oligonucleotides D8-10F and D8-13R (SEQ ID NOs:242 and243) as primers, the fourth portion of the codon-optimized Δ8 desaturasegene was amplified similarly by PCR and cloned into pGEM-T-easy vectorto generate pT8(10-13).

E. coli was transformed separately with pT8(1-3), pT8(4-6), pT8(7-9) andpT8(10-13) and the plasmid DNA was isolated from ampicillin-resistanttransformants. Plasmid DNA was purified and digested with theappropriate restriction endonucleases to liberate the 309 bp NcoI/BglIIfragment of pT8(1-3) (SEQ ID NO:244), the 321 bp BglII/XhoI fragment ofpT8(4-6) (SEQ ID NO:245), the 264 bp XhoI/SacI fragment of pT8(7-9) (SEQID NO:246) and the 369 bp Sac1/Not1 fragment of pT8(10-13) (SEQ IDNO:247). These fragments were then combined and directionally ligatedtogether with Nco1/Not1 digested pY54PC (SEQ ID NO:120; WO2004/101757)to generate pDMW240 (FIG. 8D). This resulted in a synthetic Δ8desaturase gene (“D85-1”, SEQ ID NO:209) in pDMW240.

Compared with the published Δ8 desaturase amino acid sequence (SEQ IDNO:43) of E. gracilis, the second amino acid of D8s⁻¹ was changed from‘K’ to ‘E’ in order to add the NcoI site around the translationinitiation codon. Another version of the synthesized gene, with theexact amino acid sequence as the published E. gracilis Δ8 desaturasesequence (SEQ ID NO:43), was constructed by in vitro mutagenesis(Stratagene, San Diego, Calif.) using pDMW240 as a template andoligonucleotides ODMW390 and ODMW391 (SEQ ID NOs:248 and 249) asprimers. The resulting plasmid was designated pDMW255. The synthetic Δ8desaturase gene in pDMW255 was designated as “D85-2” and the amino acidsequence was exactly the same as the sequence depicted in SEQ ID NO:43.

Nonfunctional Codon-Optimized Δ8 Desaturase Genes

Yarrowia lipolytica strain ATCC #76982(Leu-) was transformed withpDMW240 (FIG. 8D) and pDMW255, respectively, as described in the GeneralMethods. Yeast containing the recombinant constructs were grown in MMsupplemented with EDA [20:2(11,14)]. Specifically, single colonies oftransformant Y. lipolytica containing either pDMW240 (containing D8s⁻¹)or pDMW255 (containing D8S-2) were grown in 3 mL MM at 30° C. to anOD₆₀₀ ˜1.0. For substrate feeding, 100 μl of cells were then subculturedin 3 mL MM containing 10 μg of EDA substrate for about 24 hr at 30° C.The cells were collected by centrifugation, lipids were extracted, andfatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

Neither transformant produced DGLA from EDA and thus D8s⁻¹ and D8S-2were not functional and could not desaturate EDA. The chimericD8s⁻¹::XPR and D8S-2::XPR genes are shown in SEQ ID NOs:250 and 251,respectively.

A three amino acid difference between the protein sequence of the Δ8desaturase deposited in GenBank (Accession No. AAD45877 [SEQ ID NO:43])and in WO 00/34439 or Wallis et al. (Archives of Biochem. Biophys,365:307-316 (1999)) (SEQ ID NO:252 herein) was found. Specifically,three amino acids appeared to be missing in GenBank Accession No.AAD45877. Using pDMW255 as template and ODMW392 and ODMW393 (SEQ IDNOs:253 and 254) as primers, 9 bp were added into the synthetic D8S-2gene by in vitro mutagenesis (Stratagene, San Diego, Calif.), thusproducing a protein that was identical to the sequence described in WO00/34439 and Wallis et al. (supra) (SEQ ID NO:252). The resultingplasmid was called pDMW261. The synthetic Δ8 desaturase gene in pDMW261was designated as “D85-3” (SEQ ID NO:255). Following transformation ofthe pDMW261 construct into Yarrowia, a similar feeding experiment usingEDA was conducted, as described above. No desaturation of EDA to DGLAwas observed with D8S-3.

Isolation of a Euglena gracilis Δ8 Desaturase Gene

Euglena gracilis was obtained from Dr. Richard Triemer's lab at MichiganState University (East Lansing, Mich.). From 10 mL of actively growingculture, a 1 mL aliquot was transferred into 250 mL of Euglena gracilis(Eg) Medium in a 500 mL glass bottle. Eg medium was made by combining: 1g of sodium acetate, 1 g of beef extract (Catalog #U126-01, DifcoLaboratories, Detroit, Mich.), 2 g of Bacto® Tryptone (Catalog#0123-17-3, Difco Laboratories) and 2 g of Bacto® Yeast Extract (Catalog#0127-17-9, Difco Laboratories) in 970 mL of water. After filtersterilizing, 30 mL of Soil-Water Supernatant (Catalog #15-3790, CarolinaBiological Supply Company, Burlington, N.C.) was aseptically added toproduce the final Eg medium. E. gracilis cultures were grown at 23° C.with a 16 hr light, 8 hr dark cycle for 2 weeks with no agitation.

After 2 weeks, 10 mL of culture was removed for lipid analysis andcentrifuged at 1,800×g for 5 min. The pellet was washed once with waterand re-centrifuged. The resulting pellet was dried for 5 min undervacuum, resuspended in 100 μL of trimethylsulfonium hydroxide (TMSH) andincubated at room temperature for 15 min with shaking. After this, 0.5mL of hexane was added and the vials were incubated for 15 min at roomtemperature with shaking. Fatty acid methyl esters (5 μL injected fromhexane layer) were separated and quantified using a Hewlett-Packard 6890Gas Chromatograph fitted with an Omegawax 320 fused silica capillarycolumn (Catalog #24152, Supelco Inc.). The oven temperature wasprogrammed to hold at 220° C. for 2.7 min, increase to 240° C. at 20°C./min and then hold for an additional 2.3 min. Carrier gas was suppliedby a Whatman hydrogen generator. Retention times were compared to thosefor methyl esters of standards commercially available (Catalog #U-99-A,Nu-Chek Prep, Inc.) and the resulting chromatogram is shown in FIG. 9.

The remaining 2 week culture (240 mL) was pelleted by centrifugation at1,800×g for 10 min, washed once with water and re-centrifuged. Total RNAwas extracted from the resulting pellet using the RNA STAT-60™ reagent(TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer'sprotocol provided (use 5 mL of reagent, dissolved RNA in 0.5 mL ofwater). In this way, 1 mg of total RNA (2 mg/mL) was obtained from thepellet. The mRNA was isolated from 1 mg of total RNA using the mRNAPurification Kit (Amersham Biosciences, Piscataway, N.J.) following themanufacturer's protocol provided. In this way, 85 μg of mRNA wasobtained.

cDNA was synthesized from 765 ng of mRNA using the SuperScript™ ChoiceSystem for cDNA synthesis (Invitrogen™ Life Technologies, Carlsbad,Calif.) with the provided oligo(dT) primer according to themanufacturer's protocol. The synthesized cDNA was dissolved in 20 μL ofwater.

The E. gracilis Δ8 desaturase was amplified from cDNA witholigonucleotide primers Eg5-1 and Eg3-3 (SEQ ID NOs:256 and 257) usingthe conditions described below. Specifically, cDNA (1 μL) was combinedwith 50 pmol of Eg5-1, 50 pmol of Eg5-1, 1 μL of PCR nucleotide mix (10mM, Promega, Madison, Wis.), 5 μL of 10×PCR buffer (Invitrogen), 1.5 μLof MgCl₂ (50 mM, Invitrogen), 0.5 μL of Taq polymerase (Invitrogen) andwater to 50 μL. The reaction conditions were 94° C. for 3 min followedby 35 cycles of 94° C. for 45 sec, 55° C. for 45 sec and 72° C. for 1min. The PCR was finished at 72° C. for 7 min and then held at 4° C. ThePCR reaction was analyzed by agarose gel electrophoresis on 5 μL and aDNA band with molecular weight around 1.3 kB was observed. The remaining45 μL of product was separated by agarose gel electrophoresis and theDNA band was purified using the Zymoclean™ Gel DNA Recovery Kit (ZymoResearch, Orange, Calif.) following the manufacturer's protocol. Theresulting DNA was cloned into the pGEM®-T Easy Vector (Promega)following the manufacturer's protocol. Multiple clones were sequencedusing T7, M13-28Rev, Eg3-2 and Eg5-2 (SEQ ID NOS:258-261, respectively).

Thus, two classes of DNA sequences were obtained, Eg5 (SEQ ID NO:44) andEg12 (SEQ ID NO:46), that differed in only a few bp. Translation of Eg5and Eg12 gave rise to protein sequences that differed in only one aminoacid, SEQ ID NO:45 and 47, respectively. Thus, the DNA and proteinsequences for Eg5 are set forth in SEQ ID NO:44 and SEQ ID NO:45,respectively; the DNA and protein sequences for Eg12 are set forth inSEQ ID NO:46 and SEQ ID NO:47, respectively.

Comparison of the Isolated E. Gracilis Δ8 Desaturase Sequences toPublished E. gracilis Δ8 Desaturase Sequences

An alignment of the protein sequences set forth in SEQ ID NO:45 (Eg5)and SEQ ID NO:47 (Eg12) with the protein sequence from GenBank AccessionNo. AAD45877 (gi: 5639724; SEQ ID NO:43 herein) and with the publishedprotein sequences of Wallis et al. (Archives of Biochem. Biophys.,365:307-316 (1999); WO 00/34439) [SEQ ID NO:252 herein] is shown in FIG.10. Amino acids conserved among all 4 sequences are indicated with anasterisk (*). Dashes are used by the program to maximize alignment ofthe sequences. The putative cytochrome b₅ domain is underlined. Aputative His box is shown in bold. Percent identity calculationsrevealed that the Eg5 Δ8 desaturase protein sequence is 95.5% identicalto SEQ ID NO:43 and 96.2% identical to SEQ ID NO:252, wherein “%identity” is defined as the percentage of amino acids that are identicalbetween the two proteins. Sequence alignments and percent identitycalculations were performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences was performed using the Clustal method ofalignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For a more complete analysisof the differences between the various E. gracilis Δ8 desaturasesequences, refer to co-pending U.S. patent application Ser. No.11/166,993.

Functional Analysis of the Euglena gracilis Δ8 Desaturase Sequences inSaccharomyces cerevisiae

The yeast episomal plasmid (YEp)-type vector pRS425 (Christianson etal., Gene, 110:119-22 (1992)) contains sequences from the Saccharomycescerevisiae 2μ endogenous plasmid, a LEU2 selectable marker and sequencesbased on the backbone of a multifunctional phagemid, pBluescript II SK+.The S. cerevisiae strong, constitutive glyceraldehyde-3-phosphatedehydrogenase (GPD) promoter was cloned between the SacII and SpeI sitesof pRS425 in the same way as described in Jia et al. (PhysiologicalGenomics, 3:83-92 (2000)) to produce pGPD-425. A NotI site wasintroduced into the BamHI site of pGPD-425 (thus producing a NotI siteflanked by BamHI sites), thereby resulting in plasmid pY-75. Eg5 (SEQ IDNO:44) and Eg12 (SEQ ID NO:46) were released from the pGEM®-T Easyvectors described above by digestion with NotI and cloned into the NotIsite of pY-75 to produce pY89-5 (deposited as ATCC #PTA-6048) andpY89-12, respectively. In this way, the Δ8 desaturases (i.e., Eg5 [SEQID NO:44] and Eg12 [SEQ ID NO:46]) were cloned behind a strongconstitutive promoter for expression in S. cerevisiae. A map of pY89-5is shown in FIG. 8E.

Plasmids pY89-5, pY89-12 and pY-75 were transformed into Saccharomycescerevisiae BY4741 (ATCC #201388) using standard lithium acetatetransformation procedures. Transformants were selected on DOBA mediasupplemented with CSM-leu (Qbiogene, Carlsbad, Calif.). Transformantsfrom each plate were inoculated into 2 mL of DOB medium supplementedwith CSM-leu (Qbiogene) and grown for 1 day at 30° C., after which 0.5mL was transferred to the same medium supplemented with either EDA orEtrA to 1 mM. These were incubated overnight at 30° C., 250 rpm, andpellets were obtained by centrifugation and dried under vacuum. Pelletswere transesterified with 50 μL of TMSH and analyzed by GC as describedin the General Methods. Two clones for pY-75 (i.e., clones 75-1 and75-2) and pY89-5 (i.e., clones 5-6-1 and 5-6-2) were each analyzed,while two sets of clones for pY89-12 (i.e., clones 12-8-1, 12-8-2,12-9-1 and 12-9-2) from two independent transformations were analyzed.

The lipid profile obtained by GC analysis of clones fed EDA are shown inTable 19; and the lipid profile obtained by GC analysis of clones fedEtrA are shown in Table 20. Fatty acids are identified as 16:0(palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 20:2[EDA], 20:3 (8,11,14) [DGLA], 20:3 (11,14,17) [ETrA] and 20:4(8,11,14,17) [ETA]; and the composition of each is presented as a % ofthe total fatty acids.

TABLE 19 Lipid Analysis Of Transformant S. cerevisiae Overexpressing TheEuglena gracilis Δ8 Desaturases: EDA Substrate Feeding % 20:2 20:3 Con-Clone 16:0 16:1 18:0 18:1 20:2 (8, 11, 14) verted 75-1 (control) 14 32 538 10 0 0 75-2 (control) 14 31 5 41 9 0 0 5-6-1 (Eg5) 14 32 6 40 6 2 245-6-2 (Eg5) 14 30 6 41 7 2 19 12-8-1 (Eg12) 14 30 6 41 9 1 7 12-8-2(Eg12) 14 32 5 41 8 1 8 12-9-1 (Eg12) 14 31 5 40 9 1 8 12-9-2 (Eg12) 1432 5 41 8 1 7

TABLE 20 Lipid Analysis Of Transformant S. cerevisiae Overexpressing TheEuglena gracilis Δ8 Desaturases: ETrA Substrate Feeding 20:3 20:4 % 20:3(11, 14, (8, 11, Con- Clone 16:0 16:1 18:0 18:1 17) 14, 17) verted 75-1(control) 12 25 5 33 24 0 0 75-2 (control) 12 24 5 36 22 1 5 5-6-1 (Eg5)13 25 6 34 15 7 32 5-6-2 (Eg5) 13 24 6 34 17 6 27 12-8-1 (Eg12) 12 24 534 22 2 8 12-8-2 (Eg12) 12 25 5 35 20 2 9 12-9-1 (Eg12) 12 24 5 34 22 29 12-9-2 (Eg12) 12 25 6 35 20 2 9

The data in Tables 19 and 20 showed that the cloned Euglena Δ8desaturases were able to desaturate EDA and EtrA. The sequence set forthin SEQ ID NO:47 has one amino acid change compared to the sequence setforth in SEQ ID NO:45 and has reduced Δ8 desaturase activity.

The small amount of 20:4(8,11,14,17) generated by clone 75-2 in Table 20had a slightly different retention time than a standard for20:4(8,11,14,17). This peak was more likely a small amount of adifferent fatty acid generated by the wild-type yeast in thatexperiment.

Further Modification of the Δ8 Desaturase Gene Codon-Optimized forYarrowia lipolytica

The amino acid sequence of the synthetic D8S-3 gene in pDMW261 wascorrected according to the amino acid sequence of the functional EuglenaΔ8 desaturase (SEQ ID NOs:44 and 45). Using pDMW261 as a template andoligonucleotides ODMW404 (SEQ ID NO:262) and D8-13R (SEQ ID NO:243), theDNA fragment encoding the synthetic D8S-3 desaturase gene was amplified.The resulting PCR fragment was purified with Bio101's Geneclean kit andsubsequently digested with Kpn1 and Not1 (primer ODMW404 introduced aKpnI site while primer D8-13R introduced a NotI site). The Kpn1/Not1fragment (SEQ ID NO:263) was cloned into Kpn1/Not1 digested pKUNFmKF2(FIG. 11A; SEQ ID NO:121) to produce pDMW277 (FIG. 11B).

Oligonucleotides YL521 and YL522 (SEQ ID NOs:264 and 265), which weredesigned to amplify and correct the 5′ end of the D8S-3 gene, were usedas primers in another PCR reaction where pDMW277 was used as thetemplate. The primers introduced into the PCR fragment a Nco1 site andBglII site at its 5′ and 3′ ends, respectively. The 318 bp PCR productwas purified with Bio101's GeneClean kit and subsequently digested withNco1 and BglII. The digested fragment, along with the 954 bp BglII/NotIfragment from pDMW277, was used to exchange the NcoI/NotI fragment ofpZF5T-PPC (FIG. 11C; SEQ ID NO:122) to form pDMW287. In addition tocorrecting the 5′ end of the synthetic D8S-3 gene, this cloning reactionalso placed the synthetic Δ8 desaturase gene under control of theYarrowia lipolytica FBAIN promoter (SEQ ID NO:162).

The first reaction in a final series of site-directed mutagenesisreactions was then performed on pDMW287. The first set of primers, YL525and YL526 (SEQ ID NOs:266 and 267), was designed to correct amino acidfrom F to S (position #50) of the synthetic D8S-3 gene in pDMW287. Theplasmid resulting from this mutagenesis reaction then became thetemplate for the next site-directed mutagenesis reaction with primersYL527 and YL528 (SEQ ID NOs:268 and 269). These primers were designed tocorrect the amino acid from F to S (position #67) of the D8S-3 gene andresulted in creation of plasmid pDMW287/YL527.

To complete the sequence corrections within the second quarter of thegene, the following reactions were carried out concurrently with themutations on the first quarter of the gene. Using pDMW287 as templateand oligonucleotides YL529 and YL530 (SEQ ID NOs:270 and 271) asprimers, an in vitro mutagenesis reaction was carried out to correct theamino acid from C to W (position #177) of the synthetic D8S-3 gene. Theproduct (i.e., pDMW287/Y529) of this mutagenesis reaction was used asthe template in the following reaction using primers YL531 and YL532(SEQ ID NOs:272 and 273) to correct the amino acid from P to L (position#213). The product of this reaction was called pDMW287/YL529-31.

Concurrently with the mutations on the first and second quarter of thegene, reactions were similarly carried out on the 3′ end of the gene.Each subsequent mutagenesis reaction used the plasmid product from thepreceding reaction. Primers YL533 and YL534 (SEQ ID NOs:274 and 275)were used on pDMW287 to correct the amino acid from C to S (position#244) to create pDMW287/YL533. Primers YL535 and YL536 (SEQ ID NOs:276and 277) were used to correct the amino acid A to T (position #280) inthe synthetic D8S-3 gene of pDMW287/YL533 to form pDMW287/YL533-5.Finally, the amino acid P at position #333 was corrected to S in thesynthetic D8S-3 gene using pDMW287/YL533-5 as the template and YL537 andYL538 (SEQ ID NOs:278 and 279) as primers. The resulting plasmid wasnamed pDMW287/YL533-5-7.

The BglII/XhoI fragment of pDMW287/YL529-31 and the XhoI/NotI fragmentof pDMW287/YL533-5-7 were used to change the BglII/NotI fragment ofpDMW287/YL257 to produce pDMW287F (FIG. 11D) containing the completelycorrected synthetic Δ8 desaturase gene, designated D8SF and set forth inSEQ ID NO:48. SEQ ID NO:49 sets forth the amino acid sequence encoded bynucleotides 2-1270 of SEQ ID NO:48, which is essentially the same as thesequence set forth in SEQ ID NO:45, except for an additional valinefollowing the start methionine.

Example 8 Functional Expression of the Codon-Optimized Δ9 Elongase Geneand Codon-Optimized Δ8 Desaturase In Yarrowia lipolytica

The present Example describes DGLA biosynthesis and accumulation inYarrowia lipolytica that was transformed to co-express thecodon-optimized Δ9 elongase and codon-optimized Δ8 desaturase fromExamples 6 and 7. This experiment thereby confirmed both genes' activityand Y. lipolytica's ability to express the Δ9 elongase/Δ8 desaturasepathway.

Specifically, the ClaI/PacI fragment comprising a chimericFBAIN::D8SF::Pex16 gene of construct pDMW287F (Example 7) was insertedinto the ClaI/PacI sites of pDMW237 (Example 6) to generate theconstruct pDMW297 (FIG. 11E; SED ID NO:123).

Plasmid pDMW297 contained the following components:

TABLE 21 Description of Plasmid pDMW297(SEQ ID NO: 123) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:123 Components EcoRI/ClaI ARS18 sequence (GenBank Accession No.(9053-10448) A17608) ClaI/PacI FBAIN::D8SF::Pex16, comprising: (1-2590)FBAIN: FBAIN promoter (SEQ ID NO: 162) D8SF: codon-optimized Δ8desaturase gene (SEQ ID NO: 48), derived from Euglena gracilis (GenBankAccession No. AF139720) Pex16: Pex16 terminator sequence of YarrowiaPex16 gene (GenBank Accession No. U75433) PacI/SalI Yarrowia Ura3 gene(GenBank Accession No. (2590-4082) AJ306421) SalI/BsiWIFBAIN::IgD9e::Pex20, comprising: (4082-6257) FBAIN: FBAIN promoter (SEQID NO: 162) IgD9e: codon-optimized Δ9 elongase gene (SEQ ID NO: 41),derived from Isochrysis galbana (GenBank Accession No. 390174) Pex20:Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613)

Construct pDMW297 was then used for transformation of strain Y2031(Example 5) according to the General Methods. The transformant cellswere plated onto MM selection media plates and maintained at 30° C. for2 to 3 days. A total of 8 transformants grown on the MM plates werepicked and re-streaked onto fresh MM plates. Once grown, these strainswere individually inoculated into liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation, lipidswere extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that DGLA was produced in all of the transformantsanalyzed. One strain produced about 3.2%, 4 strains produced 4.3-4.5%,two strains produced 5.5-5.8% and one strain produced 6.4% DGLA(designated herein as strain “Y0489”). The “percent (%) substrateconversion” of the codon-optimized D8SF gene in strain Y0489 wasdetermined to be 75%.

Example 9 Δ9 Elongase/Δ8 Desaturase Pathway: Generation of Y2214 Strainto Produce about 14% ARA of Total Lipids

The present Example describes the construction of strain Y2214, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing 14% ARArelative to the total lipids (FIG. 4). This strain was engineered toexpress the Δ9 elongase/Δ8 desaturase pathway; thus, analysis of thecomplete lipid profiles of strain Y2214 indicating no GLA co-synthesisin the final ARA-containing oil was expected.

The development of strain Y2214 herein required the construction ofstrains Y2152 and Y2153 (producing ˜3.5% DGLA), strains Y2173 and Y2175(producing 14-16% DGLA), and strains Y2183 and 2184 (producing 5% ARA).

Generation of Strains Y2152 and Y2153 to Produce about ˜3.5% DGLA ofTotal Lipids

Construct pZP2C16M899 (FIG. 12A, SEQ ID NO:124) was used to integrate acluster of four chimeric genes (comprising two Δ9 elongases, a syntheticC_(16/18) fatty acid elongase and a Δ8 desaturase), as well as aYarrowia AHAS gene (acetohydroxy-acid synthase) containing a singleamino acid mutation. The mutated AHAS enzyme in Yarrowia conferredresistance to sulfonylurea, which was used as a positive screeningmarker. Plasmid pZP2C16M899 was designed to integrate into the Pox2 genesite of Yarrowia strain ATCC #20362 and thus contained the followingcomponents:

TABLE 22 Description of Plasmid pZP2C16M899 (SEQ ID NO: 124) RE SitesAnd Nucleotides Within SEQ Description Of Fragment And Chimeric Gene IDNO: 124 Components BsiWI/AscI 810 bp 5′ part of Yarrowia Aco2 gene(GenBank (6152-6962) Accession No. AJ001300) SphI/EcoRI 655 bp 3′ partof Yarrowia Aco2 gene (GenBank (9670-10325) Accession No. AJ001300)BsiWI/PmeI GPM/FBAintron::rELO2S::Oct, comprising: with EcoRV GPM/FBAIN:GPM::FBAIN chimeric promoter (SEQ (929-3195) ID NO: 167) rELO2S:codon-optimized rELO2 elongase gene (SEQ ID NO: 52), derived from rat(GenBank Accession No. AB071986) OCT: OCT terminator sequence ofYarrowia OCT gene (GenBank Accession No. X69988) BsiWI/EcoRIGPAT::IgD9e::Pex20, comprising: (929-14447, GPAT: GPAT promoter (SEQ IDNO: 164) reverse) IgD9e: codon-optimized Δ9 elongase gene (SEQ ID NO:41), derived from I. galbana Pex20: Pex20 terminator sequence ofYarrowia Pex20 gene (GenBank Accession No. AF054613) EcoRI/SwaITEF::IgD9e::Lip1, comprising: (14447-12912) TEF: TEF promoter (GenBankAccession No. AF054508) IgD9e: codon-optimized Δ9 elongase gene (SEQ IDNO: 41), derived from I. galbana Lip1: Lip1 terminator sequence ofYarrowia Lip1 gene (GenBank Accession No. Z50020) SwaI/PacIFBAIN::D8SF::Pex16, comprising: (12912-10325) FBAIN: FBAIN promoter (SEQID NO: 162) D8SF: codon-optimized Δ8 desaturase gene (SEQ ID NO: 48),derived from Euglena gracilis (GenBank Accession No. AF139720) Pex16:Pex16 terminator sequence of Yarrowia Pex16 gene (GenBank Accession No.U75433) gene PmeI with Yarrowia lipolytica AHAS gene comprising aEcoRV/BsiWI W497L mutation (SEQ ID NO: 280) (3195-6152)

Plasmid pZP2C16M899 was digested with SphI/AscI, and then used totransform ATCC #20362 according to the General Methods. Followingtransformation, cells were plated onto MM plates containing 150 mgsulfonylurea and maintained at 30° C. for 2 to 3 days. The sulfonylurearesistant colonies were picked and streaked onto MM with sulfonylureaselection plates. A total of 96 transformants were then inoculated intoliquid MM with sulfonylurea at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed the presence of DGLA in the transformants containingthe 4 chimeric genes of pZP2C16M899, but not in the wild type Yarrowiacontrol strain. Most of the selected 96 strains produced less than 2%DGLA of total lipids. There were 28 strains that produced 2-2.9% DGLA oftotal lipids. There were 2 strains that produced about 3.5% DGLA oftotal lipids. Strains #65 and #73 were designated herein as strains“Y2152” and “Y2153”, respectively.

Generation of Strains Y2173 and Y2175 to Produce about 14-16% DGLA ofTotal Lipids

Construct pDMW314 (FIG. 12B, SEQ ID NO:125) was used to integrate acluster of four chimeric genes (comprising two Δ9 elongases, a Δ8desaturase and a Δ12 desaturase) into the Ura3 gene site of Yarrowiastrains Y2152 and Y2153, to thereby enhance production of DGLA. PlasmidpDMW314 contained the following components:

TABLE 23 Description of Plasmid pDMW314 (SEQ ID NO: 125) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:125 Components AscI/BsiWI 784 bp 5′ part of Yarrowia Ura3 gene (GenBank(10066-9275) Accession No. AJ306421) SphI/PacI 516 bp 3′ part ofYarrowia Ura3 gene (GenBank (12774-1) Accession No. AJ306421) SwaI/BsiWIFBAIN::F.D12S::Pex20, comprising: (6582-9275) FBAIN: FBAIN promoter (SEQID NO: 162) F.Δ12: Fusarium moniliforme Δ12 desaturase gene (SEQ ID NO:27) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBankAccession No. AF054613) ClaI/EcoRI GPAT::IgD9E::Pex20: as described forpZP2C16M899 (6199-4123) (supra) EcoRI/SwaI TEF:: IgD9E::Lip1: asdescribed for pZP2C16M899 (4123-2588) (supra) SwaI/PacIFBAIN::D8SF::Pex16: as described for pZP2C16M899 (2588-1) (supra)Plasmid pDMW314 was digested with AscI/SphI, and then used fortransformation of Y. lipolytica strains Y2152 and Y2153 according to theGeneral Methods. The transformant cells were plated onto FOA selectionmedia plates and maintained at 30° C. for 2 to 3 days. The FOA resistantcolonies were picked and streaked onto MM and MMU selection plates. Thecolonies that could grow on MMU plates but not on MM plates wereselected as Ura− strains. Single colonies of Ura− strains were theninoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed increased production of DGLA in almost alltransformants containing the 4 chimeric genes of pDMW314. Most of theselected 48 Ura⁻ strains of Y2152 with pDMW314 produced about 6-8% DGLAof total lipids. There was one strain (i.e., #47, designated herein as“Y2173”) that produced about 13.9% DGLA of total lipids.

Most of the selected 24 Ura⁻ strains of Y2153 with pDMW314 producedabout 6-8% DGLA of total lipids. There were two strains (i.e., #6 and#11, designated herein as strains “Y2175” and “Y2176”) that producedabout 16.3% and 17.2% DGLA of total lipids, respectively.

Generation of Strains Y2183 and Y2184 to Produce about 5% ARA of TotalLipids

Construct pDMW322 (FIG. 12C, SEQ ID NO:126) was used to integrate acluster of two chimeric Δ5 desaturase genes into the Leu2 gene site ofYarrowia Y2173 and Y2175 strains to thereby enable production of ARA.Plasmid pDMW322 contained the following components:

TABLE 24 Description of Plasmid pDMW232 (SEQ ID NO: 126) RE Sites AndNucleotides Within SEQ ID NO: 126 Description Of Fragment And ChimericGene Components AscI/BsiWI 788 by 5′ part of Yarrowia Leu2 gene (GenBankAccession (3437-2642) No. AF260230) SphI/PacI 703 by 3′ part of YarrowiaLeu2 gene (GenBank Accession (6854-6145) No. AF260230) SwaI withFBAIN::MAΔ5::Pex20, comprising: PmeI/BsiWI FBAIN: FBAIN promoter (SEQ IDNO: 162) (1-2642) MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ IDNO: 6) (GenBank Accession No. AF067654) Pex20: Pex20 terminator sequenceof Yarrowia Pex20 gene (GenBank Accession No. AF054613) EcoRI/SwaIGPM/FBAIN::I.Δ5S::Oct, comprising: with PmeI GPM/FBAIN: GPM::FBAINchimeric promoter (SEQ ID (8833-1) NO: 167) I.Δ5S: codon-optimized Δ5desaturase gene (SEQ ID NO: 10), derived from Isochrysis galbana (WO2002/ 081668) OCT: OCT terminator sequence of Yarrowia OCT gene (GenBankAccession No. X69988) EcoRI/PmeI Yarrowia Ura3 gene (GenBank AccessionNo. AJ306421) (8833-7216)

Plasmid pDMW322 was digested with AscI/SphI, and then used to transformstrains Y2173 and Y2175 separately according to the General Methods.Following transformation, the cells were plated onto MMLe plates andmaintained at 30° C. for 2 to 3 days. The individual colonies grown onMMLe plates from each transformation were picked and streaked onto MMand MMLe plates. Those colonies that could grow on MMLe plates but noton MM plates were selected as Leu2⁻ strains. Single colonies of Leu2⁻strains were then inoculated into liquid MMLe media at 30° C. and shakenat 250 rpm/min for 2 days. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of ARA in pDMW322 transformants, but notin the parental Y2173 and Y2175 strains. Specifically, among the 48selected Leu2⁻ transformants of Y2173 with pDMW322, most strainsproduced less than 4.4% ARA of total lipids; however, there were twostrains (i.e., #1 and #42, designated herein as strains “Y2181” and“Y2182”) that produced about 4.5 and 5.8% ARA of total lipids,respectively.

In parallel, among the 48 selected Leu2⁻ transformants of Y2175 withpDMW322, most strains produced less than 4.5% ARA of total lipids. Therewere three strains (i.e., #22, #42 and #47, designated herein as strains“Y2183”, “Y2184” and “Y2185”), that produced about 4.9%, 4.6% and 4.7%ARA of total lipids, respectively, in the engineered Yarrowia.

Generation of Strain Y2214 to Produce about 14% ARA of Total Lipids

Construct pZKSL5598 (FIG. 12D, SEQ ID NO:127) was used to integrate acluster of four chimeric genes (comprising a Δ9 elongase, a Δ8desaturase and two Δ5 desaturases) into the Lys5 gene (Gen BankAccession No. M34929) site of Yarrowia Y2183 and Y2185 strains tothereby enhance production of ARA. Plasmid pZKSL5598 contained thefollowing components:

TABLE 25 Description of Plasmid pZKSL5598 (SEQ ID NO: 127) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:127 Components AscI/BsiWI 794 bp 5′ part of Yarrowia Lys5 gene (GenBank(10409-9573) Accession No. M34929) SphI/PacI 687 bp 3′ part of YarrowiaLys5 gene (GenBank (13804-13117) Accession No. M34929) BsiWI/SwaINT::I.D5S::Lip1, comprising: (7150-9573) NT: YAT1 promoter (SEQ ID NO:165) I.Δ5S: codon-optimized Δ5 desaturase gene (SEQ ID NO: 10), derivedfrom Isochrysis galbana (WO 2002/ 081668) Lip1: Lip1 terminator sequenceof Yarrowia Lip1 gene (GenBank Accession No. Z50020) SalI/BsiWIGPAT::MAΔ5::Pex20, comprising: (4537-7150) GPAT: GPAT promoter (SEQ IDNO: 164) MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ ID NO: 6)(GenBank Accession No. AF067654) Pex20: Pex20 terminator sequence fromYarrowia Pex20 gene (GenBank Accession No. AF054613) SwaI/PmeIFBAINm::IgD9e::OCT, comprising: (2381-348) FBAINm: FBAINm promoter (SEQID NO: 163) IgD9e: codon-optimized Δ9 elongase gene (SEQ ID NO: 41),derived from I. galbana OCT: OCT terminator sequence of Yarrowia OCTgene (GenBank Accession No. X69988) ClaI/PacI GPD::D8SF::Pex16,comprising: (1-13804) GPD: GPD promoter (SEQ ID NO: 158) D8SF:codon-optimized Δ8 desaturase gene (SEQ ID NO: 48), derived from Euglenagracilis (GenBank Accession No. AF139720) Pex16: Pex16 terminatorsequence of Yarrowia Pex16 gene (GenBank Accession No. U75433)SalII/PmeI Yarrowia Leu2 gene (GenBank Accession No. (4537-2417)AF260230)

Plasmid pZKSL5598 was digested with AscI/SphI, and then used totransform strains Y2183 and Y2184 separately according to the GeneralMethods. Following transformation, the cells were plated onto MMLysplates and maintained at 30° C. for 2 to 3 days. The individual coloniesgrown on MMLys plates from each transformation were picked and streakedonto MM and MMLys plates. Those colonies that could grow on MMLys platesbut not on MM plates were selected as Lyse strains. Single colonies ofLys⁻ strains were then inoculated into liquid MMLys media at 30° C. andshaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed increased production of ARA in pZKSL5598transformants. Among the 48 selected Lys⁻ transformants of Y2183 withpZKSL5598, most strains produced between 4-9.5% ARA of total lipids.Three strains (i.e., #7, #12 and #37, designated herein as strains“Y2209”, “Y2210” and “Y2211”) produced about 9.9%, 10.3% and 9.6% ARA oftotal lipids, respectively.

Among the 48 selected Lys⁻ transformants of Y2184 with pZKSL5598, moststrains produced between 4-11% ARA of total lipids. Two strains (i.e.,#3 and #22, designated herein as strains “Y2213” and “Y2214”) producedabout 11.9% and 14% ARA of total lipids, respectively.

Example 10 Generation of Intermediate Strain Y2067U, Producing 14% EPAof Total Lipids

The present Example describes the construction of strain Y2067U, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing significantconcentrations of EPA relative to the total lipids (FIG. 4). The affectof M. alpina LPAAT1, DGAT1 and DGAT2 gene over-expression and Y.lipolytica CPT1 gene over-expression were examined in this EPA producingstrain based on analysis of TAG content and/or composition, as describedin Examples 14, 15, 16 and 21, respectively (infra).

The development of strain Y2067U (producing 14% EPA) herein required theconstruction of strain M4 (producing 8% DGLA and described in Example4), strain Y2034 (producing 10% ARA and described in Example 4), strainE (producing 10% EPA), strain EU (producing 10% EPA) and strain Y2067(producing 15% EPA).

Generation of E Strain to Produce about 10% EPA of Total Lipids inEngineered Yarrowia

Construct pZP3L37 (FIG. 13A; SEQ ID NO:128) was created to integratethree synthetic Δ17 desaturase chimeric genes into the acyl-CoA oxidase3 gene of the Y2034 strain described in Example 4. The plasmid pZP3L37contained the following components:

TABLE 26 Description of Plasmid pZP3L37 (SEQ ID NO: 128) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:128 Components AscI/BsiWI 763 bp 5′ part of Yarrowia Pox3 gene (GenBank(6813-6043) Accession No. AJ001301) SphI/PacI 818 bp 3′ part of YarrowiaPox3 gene (GenBank (9521-10345) Accession No. AJ001301) ClaI/BsiWITEF::Δ17S::Pex20, comprising: (4233-6043) TEF: TEF promoter (GenBankAccession No. AF054508) Δ17S: codon-optimized Δ17 desaturase gene (SEQID NO: 16), derived from S. diclina (U.S. 2003/0196217 A1) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) ClaI/PmeI FBAIN::Δ17S::Lip2, comprising: (4233-1811) FBAIN:FBAIN promoter (SEQ ID NO: 162) Δ17S: SEQ ID NO: 16 (supra) Lip2: Lip2terminator sequence of Yarrowia Lip2 gene (GenBank Accession No.AJ012632) PmeI/SwaI Yarrowia Leu2 gene (GenBank Accession No. (1811-1)AF260230) PacI/SwaI FBAINm::Δ17S::Pex16, comprising: (10345-1) FBAINm:FBAINm promoter (SEQ ID NO: 163) Δ17S: SEQ ID NO: 16 (supra) Pex16:Pex16 terminator sequence of Yarrowia Pex16 gene (GenBank Accession No.U75433)

Plasmid pZP3L37 was digested with AscI/SphI, and then used to transformstrain Y2034 according to the General Methods. Following transformation,the cells were plated onto MM plates and maintained at 30° C. for 2 to 3days. A total of 48 transformants grown on the MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation, lipidswere extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of EPA in most of the transformants withpZP3L37, but not in the parental strain (i.e., Y2034). Among the 48selected transformants with pZP3L37, there were 18 strains that producedless than 2% EPA, 14 strains that produced 2-3% EPA, and 1 strain thatproduced about 7% EPA of total lipids in the engineered Yarrowia.

The strain that produced 7% EPA was further analyzed after culturing thestrain using “two-stage growth conditions”, as described in the GeneralMethods (i.e., 48 hrs MM, 72 hrs HGM). GC analyses showed that theengineered strain produced about 10% EPA of total lipids after thetwo-stage growth. The strain was designated as the “E” strain.

Generation of EU Strain to Produce about 10% EPA of Total Lipids withUra− Phenotype

Strain EU (Ura⁻) was created by identifying mutant cells of strain Ethat were 5-FOA resistant. Specifically, one loop of Yarrowia E straincells were inoculated into 3 mL YPD medium and grown at 30° C. withshaking at 250 rpm for 24 hrs. The culture was diluted with YPD to anOD₆₀₀ of 0.4 and then incubated for an additional 4 hrs. The culture wasplated (100 μl/plate) onto MM+FOA plates and maintained at 30° C. for 2to 3 days. A total of 16 FOA resistant colonies were picked and streakedonto MM and MM+FOA selection plates. From these, 10 colonies grew on FOAselection plates but not on MM plates and were selected as potentialUra⁻ strains.

One of these strains was used as host for transformation with pY37/F15,comprising a chimeric GPD::Fusarium moniliforme Δ15::XPR2 gene and aUra3 gene as a selection marker (FIG. 13B; SEQ ID NO:129). After threedays of selection on MM plates, hundreds of colonies had grown on theplates and there was no colony growth of the transformation control thatcarried no plasmid. This experiment confirmed that the 5-FOA resistanthost strain was Ura−, and this strain was designated as strain “EU”.Single colonies of the EU strain were then inoculated into liquid MMUadditionally containing 0.1 g/L uridine and cultured for 2 days at 30°C. with shaking at 250 rpm/min. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC. GC analyses showed that the EU strain producedabout 10% EPA of total lipids.

Generation of Y2067 Strain to Produce about 15% EPA of Total Lipids

Plasmid pKO2UF2PE (FIG. 13C; SEQ ID NO:130) was created to integrate acluster containing two chimeric genes (comprising a heterologous Δ12desaturase and a C_(18/20) elongase) and a Ura3 gene into the nativeYarrowia Δ12 desaturase gene of strain EU. Plasmid pKO2UF2PE containedthe following components:

TABLE 27 Description of Plasmid pKO2UF2PE (SEQ ID NO: 130) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:130 Components AscI/BsiWI 730 bp 5′ part of Yarrowia Δ12 desaturase gene(3382-2645) (SEQ ID NO: 23) SphI/EcoRI 556 bp 3′ part of Yarrowia Δ12desaturase gene (6090-6646) (SEQ ID NO: 23) SwaI/BsiWI/FBAINm::F.Δ12DS::Pex20, comprising: (1-2645) FBAINm: FBAINm promoter(SEQ ID NO: 163) F.Δ12: Fusarium moniliforme Δ12 desaturase gene (SEQ IDNO: 27) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBankAccession No. AF054613) SwaI/PmeI GPAT::EL1S::OCT, comprising: (1-8525)GPAT: GPAT promoter (SEQ ID NO: 164) EL1S: codon-optimized elongase 1gene (SEQ ID NO: 19), derived from Mortierella alpina (GenBank AccessionNo. AX464731) OCT: OCT terminator sequence of Yarrowia OCT gene (GenBankAccession No. X69988) EcoRI/PacI Yarrowia Ura3 gene (GenBank AccessionNo. (6646-8163) AJ306421)

Plasmid pKO2UF2PE was digested with AscI/SphI and then used to transformstrain EU according to the General Methods (although strain EU wasstreaked onto a YPD plate and grown for approximately 36 hr prior tosuspension in transformation buffer [versus 18 hrs]). Followingtransformation, cells were plated onto MM plates and maintained at 30°C. for 2 to 3 days. A total of 72 transformants grown on MM plates werepicked and re-streaked separately onto fresh MM plates. Once grown,these strains were individually inoculated into liquid MM at 30° C. andshaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of EPA in almost all of thetransformants with pKO2UF2PE. More specifically, among the 72 selectedtransformants, there were 17 strains that produced 8-9.9% EPA, 27strains that produced 10-10.9% EPA, 16 strains that produced 11-11.9%EPA, and 7 strains that produced 12-12.7% EPA of total lipids in theengineered Yarrowia. The strain that produced 12.7% EPA was furtheranalyzed by using the two-stage growth conditions, as described in theGeneral Methods (i.e., 48 hrs MM, 72 hrs HGM). GC analyses showed thatthe engineered strain produced about 15% EPA of total lipids after thetwo-stage growth. The strain was designated as strain “Y2067”.

Generation of Y2067U Strain to Produce about 14% EPA of Total Lipidswith Ura− Phenotype

In order to disrupt the Ura3 gene in strain Y2067, construct pZKUT16(FIG. 13D; SEQ ID NO:131) was created to integrate a TEF::rELO2S::Pex20chimeric gene into the Ura3 gene of strain Y2067. rELO2S is acodon-optimized rELO gene encoding a rat hepatic enzyme that elongates16:0 to 18:0 (i.e., a C_(16/18) elongase). The plasmid pZKUT16 containedthe following components:

TABLE 28 Description of Plasmid pZKUT16 (SEQ ID NO: 131) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:131 Components BsiWI/PacI 721 bp 5′ part of Yarrowia Ura3 gene (GenBank(1-721) Accession No. AJ306421) SalI/ClaI 724 bp 3′ part of YarrowiaUra3 gene (GenBank (3565-4289) Accession No. AJ306421) ClaI/BsiWITEF::rELO2S::Pex20, comprising: (4289-1) TEF: TEF promoter (GenBankAccession No. AF054508) rELO2S: codon-optimized rELO2 elongase gene (SEQID NO: 52), derived from rat (GenBank Accession No. AB071986) Pex20:Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613)

The plasmid pZKUT16 was digested with SalI/Pact, and then used totransform Y2067 strain according to the General Methods. Followingtransformation, cells were plated onto MM+5-FOA selection plates andmaintained at 30° C. for 2 to 3 days.

A total of 24 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. The strainsthat could grow on MM+5-FOA plates, but not on MM plates, were selectedas Ura− strains. A total of 10 Ura− strains were individually inoculatedinto liquid MMU media at 30° C. and grown with shaking at 250 rpm/minfor 1 day. The cells were collected by centrifugation, lipids wereextracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of 5 to 7% EPA in all of thetransformants with pZKUT16 after one day growth in MMU media. The strainthat produced 6.2% EPA was further analyzed using the two-stage growthconditions (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that theengineered strain produced about 14% EPA of total lipids. The strain wasdesignated as strain “Y2067U”.

The final genotype of this strain with respect to wildtype Yarrowialipolytica ATCC #20362 was as follows: Ura3-, Pox3-, Y.Δ12-,FBA::F.Δ12::Lip2, FBAINm::F.Δ12::Pex20, TEF::Δ6S::Lip1,FBAIN::E1S::Pex20, GPAT::E1S::Oct, TEF::E2S::Xpr, FBAIN::Δ5::Pex20,TEF::Δ5::Lip1, FBAIN::Δ17S::Lip2, FBAINm::Δ17S::Pex16, TEF::Δ17S andTEF::rELO2S::Pex20.

Example 11 Generation of Intermediate Strain Y2107U1, Producing 16% EPAof Total Lipids

The present Example describes the construction of strain Y2107U1,derived from Yarrowia lipolytica ATCC #20362, capable of producingsignificant concentrations of EPA relative to the total lipids (FIG. 4).The affect of M. alpina GPAT gene over-expression was examined in thisEPA producing strain based on analysis of TAG content and/orcomposition, as described in Example 17 (infra).

The development of strain Y2107U1 (producing 16% EPA and possessing aUra− phenotype) herein required the construction of strain M4 (producing8% DGLA and described in Example 4), strain Y2047 (producing 11% ARA anddescribed in Example 4), strain Y2048 (producing 11% EPA), strain Y2060(producing 13% EPA), strain Y2072 (producing 15% EPA), strain Y2072U1(producing 14% EPA) and Y2089 (producing 18% EPA).

Generation of Y2048 Strain to Produce about 11% EPA of Total Lipids

Construct pZP3L37 (FIG. 13A; SEQ ID NO:128; Example 10) was utilized tointegrate three synthetic Δ17 desaturase chimeric genes into theacyl-CoA oxidase 3 gene of strain Y2047 (Example 4). Specifically,plasmid pZP3L37 was digested with AscI/SphI, and then used to transformstrain Y2047 according to the General Methods. Following transformation,the cells were plated onto MM plates and maintained at 30° C. for 2 to 3days. A total of 96 transformants grown on the MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation, lipidswere extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of EPA in most of the transformants withpZP3L37, but not in the parental strain (i.e., Y2047). Among the 96selected transformants with pZP3L37, there were 20 strains that producedless than 2% EPA, 23 strains that produced 2-3% EPA, 5 strains thatproduced 3-4% EPA, and 2 strains (i.e., strain #71 and strain #94) thatproduced about 6% EPA of total lipids in the engineered Yarrowia.

Strain #71 (which produced 6% EPA) was further analyzed by culturing itin two-stage growth conditions (i.e., 48 hrs MM, 72 hrs HMG). GCanalyses showed that strain #71 produced about 11% EPA of total lipids.The strain was designated as “Y2048”.

Generation of Y2060 Strain to Produce about 13% EPA of Total Lipids withUra− Phenotype

In order to disrupt the Ura3 gene in strain Y2048, construct pZKUT16(FIG. 13D; SEQ ID NO:131; Example 10) was utilized to integrate aTEF::rELO2S::Pex20 chimeric gene into the Ura3 gene of strain Y2048.Specifically, plasmid pZKUT16 was digested with SalI/Pact, and then usedto transform strain Y2048 according to the General Methods. Followingtransformation, cells were plated onto MM+5-FOA selection plates andmaintained at 30° C. for 2 to 3 days.

A total of 40 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. Thosestrains that could grow on MM+5-FOA plates, but not on MM plates, wereselected as Ura− strains. Each of these 40 Ura− strains wereindividually inoculated into liquid MMU and grown at 30° C. with shakingat 250 rpm/min for 2 days. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that there were 14 strains that produced less than 5%EPA, 9 strains that produced 5-5.9% EPA, 15 strains that produced 6-6.9%EPA, and 7 strains that produced 7-8% EPA of total lipids after two daygrowth in MMU media. The strains that produced 7-8% EPA were furtheranalyzed using two-stage growth conditions (i.e., 48 hrs MM, 96 hrsHGM). GC analyses showed that all these strains produced more than 10%EPA; and, one of them produced about 13% EPA of the total lipids. Thatstrain was designated as strain “Y2060”.

Generation of Y2072 Strain to Produce about 15% EPA of Total Lipids

Construct pKO2UM25E (FIG. 14A; SEQ ID NO:132) was used to integrate acluster of three chimeric genes (comprising a C_(18/20) elongase, a Δ12desaturase and a Δ5 desaturase) and a Ura3 gene into the native YarrowiaΔ12 desaturase gene site of strain Y2060. Plasmid pKO2UM25E containedthe following components:

TABLE 29 Description of Plasmid pKO2UM25E (SEQ ID NO: 132) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:132 Components HindIII/AscI 728 bp 5′ part of Yarrowia Δ12 desaturasegene (1-728) (SEQ ID NO: 23) SphI/EcoRI 556 bp 3′ part of Yarrowia Δ12desaturase gene (3436-3992) (SEQ ID NO: 23) BsiWI/HindIIIGPAT::EL1S::XPR, comprising: (10437-1) GPAT: GPAT promoter (SEQ ID NO:164) EL1S: codon-optimized elongase 1 gene (SEQ ID NO: 19), derived fromMortierella alpina (GenBank Accession No. AX464731) XPR: ~100 bp of the3′ region of the Yarrowia Xpr gene (GenBank Accession No. M17741)BglII/BsiWI FBAIN::M.Δ12::Pex20, comprising: (7920-10437) FBAIN: FBAINpromoter (SEQ ID NO: 162) M.Δ12: Mortierella isabellina Δ12 desaturasegene (GenBank Accession No. AF417245; SEQ ID NO: 25) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) SalI/PacI Yarrowia Ura3 gene (Gene Bank Accession No.(6046-7544) AJ306421) EcoRI/SalI TEF::I.Δ5S::Pex20, comprising:(3992-6046) TEF: TEF promoter (GenBank Accession No. AF054508) I.Δ5S:codon-optimized Δ5 desaturase gene (SEQ ID NO: 10), derived fromIsochrysis galbana (WO 2002/081668) Pex20: Pex20 terminator sequence ofYarrowia Pex20 gene (GenBank Accession No. AF054613)

Plasmid pKO2UM25E was digested with SphI/AscI, and then used totransform Y2060 according to the General Methods. Followingtransformation, cells were plated onto MM plates and maintained at 30°C. for 2 to 3 days.

A total of 63 transformants grown on MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM at 30° C. and cultured withshaking at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of EPA in almost all transformants withpKO2UM25E after one-day growth in MM media. Among the 63 selectedtransformants, there were 26 strains that produced 6-8.9% EPA and 46strains that produced more than 9% EPA. The strains that produced morethan 9% EPA were selected for further analysis using two-stage growthconditions (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that 45 outof the 46 selected strains produced 11-14.5% EPA while culture #2produced 15.1% EPA of total lipids after the two-stage growth. Thisstrain (i.e., #2) was designated as strain “Y2072”.

Generation of Y2072U1 Strain to Produce about 14% EPA of Total Lipidswith Ura− Phenotype

The construct pZKUGPI5S (FIG. 14B; SEQ ID NO:133) was created tointegrate a GPAT::I.Δ5S::Pex20 chimeric gene into the Ura3 gene of Y2072strain. More specifically, plasmid pZKUGPI5S contained the followingcomponents:

TABLE 30 Description of Plasmid pZKUGPI5S (SEQ ID NO: 133) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:133 Components BsiWI/PacI 721 bp 5′ part of Yarrowia Ura3 gene (GenBank(318-1038) Accession No. AJ306421) SalI/ClaI 724 bp 3′ part of YarrowiaUra3 gene (GenBank (3882-4606) Accession No. AJ306421) ClaI/BsiWIGPAT::I.Δ5S::Pex20, comprising: (4606-318) GPAT: GPAT promoter (SEQ IDNO: 164) I.Δ5S: codon-optimized Δ5 desaturase gene (SEQ ID NO: 10),derived from Isochrysis galbana (WO 2002/081668) Pex20: Pex20 terminatorsequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613)

Plasmid pZKUGPI5S was digested with SalI/Pact, and then used totransform strain Y2072 according to the General Methods. Followingtransformation, cells were plated onto MM+5-FOA selection plates andmaintained at 30° C. for 3 to 4 days.

A total of 24 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. Thosestrains that could grow on MM+5-FOA plates, but not on MM plates, wereselected as Ura− strains. Each of these 24 Ura− strains wereindividually inoculated into liquid MMU and grown at 30° C. with shakingat 250 rpm/min for 2 days. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that there were 8 strains that produced 7.3-8.9% EPA,14 strains that produced 9-9.9% EPA, 1 strain that produced 10.5% EPA(i.e., #1) and 1 strain that produced 10.7% EPA (i.e., #23) of totallipids after two day growth in MMU. Strains #1 and #23 were furtheranalyzed using two-stage growth conditions (i.e., 48 hrs MM, 96 hrsHGM). GC analyses showed that these two strains produced about 14% EPAof total lipids after the two-stage growth. Strain #1 was designated asstrain “Y2072U1”.

Generation of Y2089 Strain to Produce about 18% EPA of Total Lipids

Construct pDMW302T16 (FIG. 14C; SEQ ID NO:134) was created to integratea cluster of four chimeric genes (comprising a C_(16/18) elongase, aC_(18/20) elongase, a Δ6 desaturase and a Δ12 desaturase) and a Ura3gene into the Yarrowia lipase1 gene site of Y2072U1 strain. PlasmidpDMW302T16 contained the following components:

TABLE 31 Description of Plasmid pDMW302T16 (SEQ ID NO: 134) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:134 Components BsiWI/AscI 817 bp 5′ part of Yarrowia lipase1 gene(GenBank (1-817) Accession No. Z50020) SphI/PacI 769 bp 3′ part ofYarrowia lipase1 gene (GenBank 3525-4294 Accession No. Z50020)EcoRI/BsiWI TEF::rELO2S::Pex20, comprising: (13328-1) TEF: TEF promoter(GenBank Accession No. AF054508) rELO2S: codon-optimized rELO2 elongasegene (SEQ ID NO: 52), derived from rat (GenBank Accession No. AB071986)Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBankAccession No. AF054613) BglII/EcoRI FBAIN::D6S::Lip1, comprising:(10599-13306) FBAIN: FBAIN promoter (SEQ ID NO: 162) Δ6S:codon-optimized Δ6 desaturase gene (SEQ ID NO: 3), derived fromMortierella alpina (GenBank Accession No. AF465281) Lip1: Lip1terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.Z50020) ClaI/PmeI GPDIN::EL1S::Lip2, comprising: (8078-10555) GPDIN:GPDIN promoter (SEQ ID NO: 159) EL1S: codon-optimized elongase 1 gene(SEQ ID NO: 19), derived from Mortierella alpina (GenBank Accession No.AX464731) Lip2: Lip2 terminator of Yarrowia lipase2 gene (GenBankAccession No. AJ012632) EcoRI/ClaI Yarrowia Ura 3 gene (Gene BankAccession No. (6450-8078) AJ306421) PacI/EcoRI TEF:: F.Δ12::Pex16,comprising: (4294-6450) TEF: TEF promoter (GenBank Accession No.AF054508) F.Δ12: Fusarium moniliforme Δ12 desaturase gene (SEQ ID NO:27) Pex16: Pex16 terminator of Yarrowia Pex16 gene (GenBank AccessionNo. U75433)

Plasmid pDMW302T16 was digested with SphI/AscI, and then used totransform strain Y2072U1 according to the General Methods. Followingtransformation, cells were plated onto MM plates and maintained at 30°C. for 3 to 4 days. A total of 48 transformants grown on MM plates werepicked and re-streaked onto fresh MM plates. Once grown, these strainswere individually inoculated into liquid MM and grown at 30° C. withshaking at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed that EPA was produced in almost all transformants ofY2072U1 with pDMW302T16 after two-day growth in MM media. Among the 48selected transformants, there were 27 strains that produced less than10% EPA, 14 strains that produced 10-12.9% EPA and 5 strains thatproduced 13-13.9% EPA. Strain #34 (producing 13.9% EPA) was selected forfurther analysis using the two-stage growth procedure (i.e., 48 hrs MM,96 hrs HGM). GC analyses showed that strain #34 produced about 18% EPAof total lipids. Strain #34 was designated as strain “Y2089”.

The genotype of strain Y2089 with respect to wildtype Yarrowialipolytica ATCC #20362 was as follows: Pox3-, LIP1-, Y.Δ12-,FBA::F.Δ12::Lip2, TEF::F.Δ12::Pex16, FBAIN::MΔ12::Pex20, TEF::Δ6S::Lip1,FBAIN::Δ6S::Lip1, FBAIN::E1S::Pex20, GPAT::E1S::Oct, GPDIN::E1S::Lip2,TEF::E2S::Xpr, FBAIN::MAΔ5::Pex20, TEF::MAΔ5::Lip1, TEF::HΔ5S::Pex16,TEF::IΔ5S::Pex20, GPAT::IΔ5S::Pex20, FBAIN::Δ17S::Lip2,FBAINm::Δ17S::Pex16, TEF::Δ17S::Pex16 and 2×TEF::rELO2S::Pex20.

Generation of Y2107U1 Strain to Produce about 16% EPA of Total Lipidswith Ura− Phenotype

Construct pZKUGPE1S (FIG. 14D; SEQ ID NO:135) was created to integrate aGPAT::EL1S::Pex20 chimeric gene into the Ura3 gene of strain Y2089. Morespecifically, plasmid pZKUGPE1S contained the following components:

TABLE 32 Description of Plasmid pZKUGPE1S (SEQ ID NO: 135) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:135 Components BsiWI/PacI 721 bp 5′ part of Yarrowia Ura3 gene (GenBank(318-1038) Accession No. AJ306421) SalI/ClaI 724 bp 3′ part of YarrowiaUra3 gene (GenBank (3882-4606) Accession No. AJ306421) ClaI/BsiWIGPAT::E1S::Pex20, comprising: (4606-318) GPAT: GPAT promoter (SEQ ID NO:164) EL1S: codon-optimized elongase 1 gene (SEQ ID NO: 19), derived fromMortierella alpina (GenBank Accession No. AX464731) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613)

Plasmid pZKUGPE1S was digested with PstI/PacI, and then used totransform strain Y2089 according to the General Methods. Followingtransformation, cells were plated onto MM+5-FOA selection plates andmaintained at 30° C. for 3 to 4 days.

A total of 8 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. Thosestrains that could grow on MM+5-FOA plates, but not on MM plates, wereselected as Ura− strains. Each of these 8 Ura− strains were individuallyinoculated into liquid MMU and grown at 30° C. with shaking at 250rpm/min for 2 days. The cells were collected by centrifugation, lipidswere extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that there were 6 strains that produced 6.6-8.7% EPAand 2 strains that produced 9.4-10% EPA (i.e., #4 and #5) of totallipids after two day growth in MMU. Strains #4 and #5 were furtheranalyzed using the two-stage growth conditions (i.e., 48 hrs MM, 96 hrsHGM). GC analyses showed that these two strains produced about 16% EPAof total lipids after the two-stage growth. Strain #4 was designated asstrain “Y2107U1” and strain #5 was designated as strain “Y2107U2”.

Example 12 Generation of Intermediate Strain MU, Producing 9-12% EPA ofTotal Lipids

The present Example describes the construction of strain MU, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing significantconcentrations of EPA relative to the total lipids (FIG. 4). The affectof various native Y. lipolytica acyltransferase knockouts were examinedin this EPA producing strain based on analysis of TAG content and/orcomposition, as described in Example 24 (infra).

The development of strain MU (producing 9-12% EPA herein) required theconstruction of strain M4 (producing 8% DGLA and described in Example4), strain Y2034 (producing 10% ARA and described in Example 4), strainE (producing 10% EPA and described in Example 10), strain EU (producing10% EPA and described in Example 10) and strain M26 (producing 14% EPA).

Generation of M26 Strain to Produce about 14% EPA of Total Lipids

Construct pKO2UM26E (SEQ ID NO:136; FIG. 15A) was used to integrate acluster of three chimeric genes (comprising a C_(18/20) elongase, a Δ6desaturase and a Δ12 desaturase) and a Ura3 gene into the Yarrowia Δ12desaturase gene site of EU strain (Example 10). Plasmid pKO2UM26Econtained the following components:

TABLE 33 Description of Plasmid pKO2UM26E (SEQ ID NO: 136) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:136 Components HindIII/AscI 728 bp 5′ part of Yarrowia Δ12 desaturasegene (1-728) (SEQ ID NO: 23) SphI/EcoRI 556 bp 3′ part of Yarrowia Δ12desaturase gene (3436-3992) (SEQ ID NO: 23) BsiWI/HindIIIGPAT::EL1S::XPR, comprising: (11095-1) GPAT: GPAT promoter (SEQ ID NO:164) EL1S: codon-optimized elongase 1 gene (SEQ ID NO: 19), derived fromMortierella alpina (GenBank Accession No. AX464731) XPR: ~100 bp of the3′ region of the Yarrowia Xpr gene (GenBank Accession No. M17741)BglII/BsiWI FBAIN::M.Δ12::Pex20, comprising: (8578-11095) FBAIN: FBAINpromoter (SEQ ID NO: 162) M.Δ12: Mortieralla isabellina Δ12 desaturasegene (GenBank Accession No. AF417245; SEQ ID NO: 25) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) SalI/PacI Yarrowia Ura3 gene (GenBank Accession No.(6704-8202) AJ306421) EcoRI/SalI FBAIN::M.Δ6B::Pex20, comprising:(3992-6704) TEF: TEF promoter (GenBank Accession No. AF054508) M.Δ6B:Mortieralla alpina Δ6 desaturase gene “B” (GenBank Accession No.AB070555; SEQ ID NO: 4) Pex20: Pex20 terminator sequence of YarrowiaPex20 gene (GenBank Accession No. AF054613)

The plasmid pKO2UM26E was digested with SphI/AscI, and then used totransform EU strain (Example 10) according to the General Methods.Following transformation, cells were plated onto MM plates andmaintained at 30° C. for 2 to 3 days.

A total of 48 transformants grown on MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM at 30° C. and grown with shakingat 250 rpm/min for 1 day. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that EPA was produced in almost all transformantswith pKO2UM26E after one-day growth in MM media. Among the 48 selectedtransformants, 5 strains produced less than 4% EPA, 23 strains produced4-5.9% EPA, 9 strains produced 6-6.9% EPA and 11 strains produced 7-8.2%EPA of total lipids in the engineered Yarrowia. The strain that produced8.2% EPA was selected for further analysis using the two-stage growthprocedure (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that theengineered strain produced about 14% EPA of total lipids. The strain wasdesignated as strain “M26”.

The genotype of the M26 strain with respect to wildtype Yarrowialipolytica ATCC #20362 was as follows: Pox3-, Y.Δ12-, FBA::F.Δ12::Lip2,FBAIN::MΔ12::Pex20, TEF::Δ6S::Lip1, FBAIN::Δ6B::Pex20,FBAIN::E1S::Pex20, GPAT::E1S::Xpr, TEF::E2S::Xpr, FBAIN::MAΔ5::Pex20,TEF::MAΔ5::Lip1, TEF::HΔ5S::Pex16, FBAIN::Δ17S::Lip2,FBAINm::Δ17S::Pex16, TEF::Δ17S::Pex16 and TEF::rELO2S::Pex20.

Generation of MU Strain to Produce about 14% EPA of Total Lipids

Strain MU was a Ura auxotroph of strain M26. This strain was made bytransforming strain M26 with 5 μg of plasmid pZKUM (SEQ ID NO:137) thathad been digested with PacI and HincII. Transformation was performedusing the Frozen-EZ Yeast Transformation kit (Zymo Research Corporation,Orange, Calif.) and transformants were selected by plating 100 μl of thetransformed cell mix on an agar plate with the following medium: 6.7 g/Lyeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.), 20 g/Ldextrose, 50 mg/L uracil and 800 mg/L FOA. After 7 days, small coloniesappeared that were plated on MM and MMU agar plates. All were URAauxotrophs. One of the strains was designated “MU”.

Example 13 Preparation of Mortierella alpina Genomic DNA and cDNA

The present Example describes the preparation of genomic DNA and cDNAfrom Mortierella alpina (ATCC #16266). This enabled isolation of the M.alpina LPAAT2, DGAT1, DGAT2, GPAT and ELO3, as described in Examples 14,15, 16, 17 and 18, respectively.

Preparation of Genomic DNA from Mortierella alpina

Genomic DNA was isolated from Mortierella alpina (ATCC #16266) using aQiaPrep Spin Miniprep Kit (Qiagen, Catalog #627106). Cells grown on aYPD agar plate (2% Bacto-yeast extract, 3% Bacto-peptone, 2% glucose,2.5% bacto-agar) were scraped off and resuspended in 1.2 mL of kitbuffer P1. The resuspended cells were placed in two 2.0 mL screw captubes, each containing 0.6 mL glass beads (0.5 mm diameter). The cellswere homogenized at the HOMOGENIZE setting on a Biospec (Bartlesville,Okla.) mini bead beater for 2 min. The tubes were then centrifuged at14,000 rpm in an Eppendorf microfuge for 2 min. The supernatant (0.75mL) was transferred to three 1.5 mL microfuge tubes. Equal volumes ofkit buffer P2 were added to each tube. After mixing the tubes byinversion three times, 0.35 mL of buffer N3 was added to each tube. Thecontents of each tube were again mixed by inversion for a total of fivetimes. The mixture was centrifuged at 14,000 rpm in an Eppendorfmicrofuge for 5 min. The supernatant from each tube was transferredindividually into 3 separate kit spin columns. The columns were thensubjected to the following steps: centrifugation (1 min at 14,000 rpm),wash once with buffer PE, centrifugation (1 min at 14,000 rpm), and thena final centrifugation (1 min at 14,000 rpm). Buffer EB (50 μl) wasadded to each column and let stand for 1 min. The genomic DNA was theneluted by centrifugation at 14,000 rpm for 1 min.

Preparation of cDNA from Mortierella alpina

cDNA of Mortierella alpina was prepared using the BD-Clontech CreatorSmart® cDNA library kit (Mississauga, ON, Canada), according to themanufacturer's protocol. Specifically, M. alpina strain ATCC #16266 wasgrown in 60 mL YPD medium (2% Bacto-yeast extract, 3% Bactor-peptone, 2%glucose) for 3 days at 23° C. Cells were pelleted by centrifugation at3750 rpm in a Beckman GH3.8 rotor for 10 min and resuspended in 6×0.6 mLTrizole reagent (Invitrogen). Resuspended cells were transferred to six2 mL screw cap tubes each containing 0.6 mL of 0.5 mm glass beads. Thecells were homogenized at the HOMOGENIZE setting on a Biospec mini beadbeater for 2 min. The tubes were briefly spun to settle the beads.Liquid was transferred to 4 fresh 1.5 mL microfuge tubes and 0.2 mLchloroform/isoamyl alcohol (24:1) was added to each tube. The tubes wereshaken by hand for 1 min and let stand for 3 min. The tubes were thenspun at 14,000 rpm for 10 min at 4° C. The upper layer was transferredto 4 new tubes. Isopropyl alcohol (0.5 mL) was added to each tube. Tubeswere incubated at room temperature for 15 min, followed bycentrifugation at 14,000 rpm and 4° C. for 10 min. The pellets werewashed with 1 mL each of 75% ethanol, made with RNase-free water andair-dried. The total RNA sample was then redissolved in 500 μl of water,and the amount of RNA was measured by A260 nm using 1:50 diluted RNAsample. A total of 3.14 mg RNA was obtained.

This total RNA sample was further purified with the Qiagen RNeasy totalRNA Midi kit following the manufacturer's protocol. Thus, the total RNAsample was diluted to 2 mL and mixed with 8 mL of buffer RLT with 80 μlof β-mercaptoethanol and 5.6 mL 100% ethanol. The sample was dividedinto 4 portions and loaded onto 4 RNeasy midid columns. The columns werethen centrifuged for 5 min at 4500×g. To wash the columns, 2 mL ofbuffer RPE was loaded and the columns centrifuged for 2 min at 4500×g.The washing step was repeated once, except that the centrifugation timewas extended to 5 min. Total RNA was eluted by applying 250 μl of RNasefree water to each column, waiting for 1 min and centrifuging at 4500×gfor 3 min.

PolyA(+)RNA was then isolated from the above total RNA sample, followingthe protocol of Amersham Biosciences' mRNA Purification Kit. Briefly, 2oligo-dT-cellulose columns were used. The columns were washed twice with1 mL each of high salt buffer. The total RNA sample from the previousstep was diluted to 2 mL total volume and adjusted to 10 mM Tris/HCl, pH8.0, 1 mM EDTA. The sample was heated at 65° C. for 5 min, then placedon ice. Sample buffer (0.4 mL) was added and the sample was then loadedonto the two oligo-dT-cellulose columns under gravity feed. The columnswere centrifuged at 350×g for 2 min, washed 2× with 0.25 mL each of highsalt buffer, each time followed by centrifugation at 350×g for 2 min.The columns were further washed 3 times with low salt buffer, followingthe same centrifugation routine. Poly(A)+RNA was eluted by washing thecolumn 4 times with 0.25 mL each of elution buffer preheated to 65° C.,followed by the same centrifugation procedure. The entire purificationprocess was repeated once. Purified poly(A)+RNA was obtained with aconcentration of 30.4 ng/μl.

cDNA was generated, using the LD-PCR method specified by BD-Clontech and0.1 μg of polyA(+) RNA sample. Specifically, for 1^(st) strand cDNAsynthesis, 3 μl of the poly(A)+RNA sample was mixed with 1 μl of SMARTIV oligo nucleotide (SEQ ID NO:281) and 1 μl of CDSIII/3′ PCR primer(SEQ ID NO:282). The mixture was heated at 72° C. for 2 min and cooledon ice for 2 min. To the tube was added the following: 2 μl first strandbuffer, 1 μl 20 mM DTT, 1 μl 10 mM dNTP mix and 1 μl Powerscript reversetranscriptase. The mixture was incubated at 42° C. for 1 hr and cooledon ice.

The 1^(st) strand cDNA synthesis mixture was used as template for thePCR reaction. Specifically, the reaction mixture contained thefollowing: 2 μl of the 1^(st) strand cDNA mixture, 2 μl 5′-PCR primer(SEQ ID NO:283), 2 μl CDSIII/3′-PCR primer (SEQ ID NO:282), 80 μl water,10 μl 10× Advantage 2 PCR buffer, 2 μl 50×dNTP mix and 2 μl 50×Advantage 2 polymerase mix. The thermocycler conditions were set for 95°C. for 20 sec, followed by 14-20 cycles of 95° C. for 5 sec and 68° C.for 6 min on a GenAmp 9600 instrument. PCR product was quantitated byagarose gel electrophoresis and ethidium bromide staining.

Seventy-five μl of the above PCR products (cDNA) were mixed with 3 μl of20 μg/μl proteinase K supplied with the kit. The mixture was incubatedat 45° C. for 20 min, then 75 μl of water was added and the mixture wasextracted with 150 μl phenol:chloroform:isoamyl alcohol mixture(25:24:1). The aqueous phase was further extracted with 150 μlchloroform:isoamyl alcohol (25:1). The aqueous phase was then mixed with15 μl of 3 M sodium acetate, 2 μl of 20 μg/μl glycogen and 400 μl of100% ethanol. The mixture was immediately centrifuged at roomtemperature for 20 min at 14000 rpm in a microfuge. The pellet waswashed once with 150 μl of 80% ethanol, air dried and dissolved in 79 μlof water.

Dissolved cDNA was subsequently digested with SfiI (79 μl of the cDNAwas mixed with 10 μl of 10×SfiI buffer, 10 μl of SfiI enzyme and 1 μl of100×BSA and the mixture was incubated at 50° C. for 2 hrs). Xylenecyanol dye (2 μl of 1%) was added. The mixture was then fractionated onthe Chroma Spin-400 column provided with the kit, following themanufacturer's procedure exactly. Fractions collected from the columnwere analyzed by agarose gel electrophoresis. The first three fractionscontaining cDNA were pooled and cDNA precipitated with ethanol. Theprecipitated cDNA was redissolved in 7 μl of water, and ligated intokit-supplied pDNR-LIB.

Library Sequencing

The ligation products were used to transform E. coli XL-1Blueelectroporation competent cells (Stratagene). An estimated total of2×10⁶ colonies was obtained. Sequencing of the cDNA library was carriedout by Agencourt Bioscience Corporation (Beverly, Mass.), using an M13forward primer (SEQ ID NO:284).

Example 14 Mortierella alpina LPAAT2 Expression Increases Percent PUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2067U (Example 10) that wastransformed to co-express the M. alpina LPAAT2 (SEQ ID NOs:69 and 70).It is contemplated art that a Y. lipolytica host strain engineered toproduce ARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9elongase/Δ8 desaturase pathway could demonstrate increased ARAbiosynthesis and accumulation, if the M. alpina LPAAT2 was similarlyco-expressed therein (e.g., in strains Y2034, Y2047 and/or Y2214).

The M. alpina LPAAT2 ORF was cloned as follows. Primers MLPAT-F andMLPAT-R (SEQ ID NO:285 and 286) were used to amplify the LPAAT2 ORF fromthe cDNA of M. alpina (Example 13) by PCR. The reaction mixturecontained 1 μl of the cDNA, 1 μl each of the primers, 22 μl water and 25μl ExTaq premix 2×Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga,520-2193, Japan). Amplification was carried out as follows: initialdenaturation at 94° C. for 150 sec, followed by 30 cycles ofdenaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec andelongation at 72° C. for 90 sec. A final elongation cycle at 72° C. for10 min was carried out, followed by reaction termination at 4° C. A ˜950bp DNA fragment was obtained from the PCR reaction. It was purifiedusing a Qiagen (Valencia, Calif.) PCR purification kit according to themanufacturer's protocol. The purified PCR product was digested with NcoIand NotI, and cloned into Nco I-Not I cut pZUF17 vector (SEQ ID NO:118;FIG. 8B), such that the gene was under the control of the Y. lipolyticaFBAIN promoter and the PEX20-3′ terminator region in theauto-replicating vector for expression in Y. lipolytica. Correcttransformants were confirmed by restriction analysis of miniprep DNA andthe resultant plasmid was designated as “pMLPAT-17” (SEQ ID NO:138).

To integrate the M. alpina LPAAT2 into the genome of Yarrowialipolytica, plasmid pMLPAT-Int was created. Primers LPAT-Re-5-1 andLPAT-Re-5-2 (SEQ ID NOs:287 and 288) were used to amplify a 1129 bp DNAfragment, YLPAT-5′ (SEQ ID NO:289), containing a 1103 bp fragment of Y.lipolytica genome immediately upstream of the AUG of the Y. lipolyticaLPAAT1 (SEQ ID NO:71). The reaction mixture contained 1 μl of Y.lipolytica genomic DNA, 1 μl each of the primers, 22 μl water and 25 μlExTaq premix 2×Taq PCR solution (TaKaRa). Amplification was carried outas described above. A ˜1130 bp DNA fragment was obtained from the PCRreaction. It was purified using Qiagen's PCR purification kit accordingto the manufacturer's protocol. The purified PCR product was digestedwith SalI and ClaI, and cloned into SalI-ClaI cut pBluescript SK (−)vector, resulting in plasmid “pYLPAT-5”.

Primers LPAT-Re-3-1 and LPAT-Re-3-2 (SEQ ID NOs:290 and 291) were thenused to amplify a 938 bp fragment, YLPAT-3′ (SEQ ID NO:292), containinga 903 bp fragment of Y. lipolytica genome immediately after the stopcodon of Y. lipolytica LPAAT1, using the same conditions as above. Thepurified PCR product was digested with ClaI and XhoI, and cloned intoClaI XhoI digested pYLPAT-5′. Correct transformants were confirmed byminiprep analysis and the resultant plasmid was designated“pYLPAT-5′-3”.

pMLPAT-17 (SEQ ID NO:138) was then digested with ClaI and NotI, and a˜3.5 kb fragment containing the Y. lipolytica URA3 gene, the Y.lipolytica FBAIN promoter and the M. alpina LPAAT2 gene was isolatedusing a Qiagen Qiaexll gel purification kit according to themanufacturer's protocol. This fragment was cloned into ClaI-NotIdigested pYLPAT-5′-3′. Correct transformants were confirmed by miniprepand restriction analysis. The resulting plasmid was named “pMLPAT-Int”(SEQ ID NO:139).

“Control” vector pZUF-MOD-1 (SEQ ID NO:140; FIG. 15B) was prepared asfollows. First, primers pzuf-mod 1 and pzuf-mod 2 (SEQ ID NOs:293 and294) were used to amplify a 252 bp “stuffer” DNA fragment using pDNR-LIB(ClonTech, Palo Alto, Calif.) as template. The amplified fragment waspurified with a Qiagen QiaQuick PCR purification kit, digested with NcoIand NotI using standard conditions, and then purified again with aQiaQuick PCR purification kit. This fragment was ligated into similarlydigested NcoI-/NotI-cut pZUF17 vector (SEQ ID NO:118; FIG. 8B) and theresulting ligation mixture was used to transform E. coli Top10 cells(Invitrogen). Plasmid DNA was purified from 4 resulting colonies, usinga Qiagen QiaPrep Spin Miniprep kit. The purified plasmids were digestedwith NcoI and NotI to confirm the presence of the ˜250 bp fragment. Theresulting plasmid was named “pZUF-MOD-1” (SEQ ID NO:140).

Y. lipolytica strain Y2067U (from Example 10, producing 14% EPA of totallipids) was transformed with plasmid pMLPAT-17, plasmid pZUF-MOD-1(control) and SpeI/XbaI digested plasmid pMLPAT-Int, individually,according to the General Methods. Transformants were grown for 2 days insynthetic MM supplemented with amino acids, followed by 4 days in HGM.The fatty acid profile of two transformants containing pZUF-MOD-1, twotransformants containing pMLPAT-17, and two transformants havingpMLPAT-Int integrated into the genome are shown below in the Table,based on GC analysis (as described in the General Methods). Fatty acidsare identified as 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA,ETA and EPA; and the composition of each is presented as a % of thetotal fatty acids.

TABLE 34 Lipid Composition In Yarrowia Strain Y2067U Engineered ToOverexpress M. alpina LPAAT2 Total Fatty Acids Strain 18:0 18:1 18:2 GLADGLA ARA ETA EPA Y2067U + pZUF-MOD-1 #1 1.1 4.7 10.9 19.4 6.3 0.9 3.913.8 Y2067U + pZUF-MOD-1 #2 0.9 4.4 9.5 19.3 6.6 0.9 4.0 14.1 Y2067U +pMLPAT-17 #1 1.0 4.4 9.8 18.6 5.9 0.8 3.4 15.5 Y2067U + pMLPAT-17 #2 0.73.5 8.4 16.7 6.2 1.0 2.9 16.0 Y2067U + pMLPAT-Int #1 1.9 4.9 13.9 21.14.8 1.1 2.7 16.6 Y2067U + pMLPAT-Int #2 1.7 4.2 12.1 21.3 5.2 1.2 2.917.3

As demonstrated above, expression of the M. alpina LPAAT2 from pMLPAT-17increased the % EPA from ˜14% in the “control” strains to 15.5-16%. Anadditional increase in EPA to 16.6-17.3% was achieved when M. alpinaLPAAT2 was integrated into the genome with pMLPAT-Int. Further increasewould be expected, if the native Yarrowia lipolytica LPAAT1 (SEQ IDNOs:71 and 72) and/or LPAAT2 (SEQ ID NOs:74 and 75) were knocked-out ine.g., strain Y2067U+pMLPAT-Int.

Example 15 Mortierella alpina DGAT1 Expression Increases Percent PUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2067U (Example 10) that wastransformed to co-express the M. alpina DGAT1 cDNA (SEQ ID NO:83). It iscontemplated that a Y. lipolytica host strain engineered to produce ARAvia either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8desaturase pathway could demonstrate increased ARA biosynthesis andaccumulation, if the M. alpina DGAT1 was similarly co-expressed therein(e.g., in strains Y2034, Y2047 and/or Y2214).

The M. alpina DGAT1 ORF was cloned as follows. First, to aid the cloningof the cDNA, the sequence of the second codon of the DGAT1 was changedfrom ‘ACA’ to ‘GCA’, resulting in an amino acid change of threonine toalanine. This was accomplished by amplifying the complete coding regionof the M. alpina DGAT1 ORF with primers MACAT-F1 and MACAT-R (SEQ IDNOs:295 and 296). Specifically, the PCR reaction mixture contained 1 μleach of a 20 μM solution of primers MACAT-F1 and MACAT-R, 1 μl of M.alpina cDNA (supra, Example 13), 22 μl water and 25 μl ExTaq premix2×Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga, 520-2193, Japan).Amplification was carried out as follows: initial denaturation at 94° C.for 150 sec, followed by 30 cycles of denaturation at 94° C. for 30 sec,annealing at 55° C. for 30 sec, and elongation at 72° C. for 90 sec. Afinal elongation cycle at 72° C. for 10 min was carried out, followed byreaction termination at 4° C. A ˜1600 bp DNA fragment was obtained fromthe PCR reaction. It was purified using Qiagen's PCR purification kitaccording to the manufacturer's protocol.

The M. alpina DGAT1 ORF was to be inserted into Nco I- and NotI-digested plasmid pZUF17 (SEQ ID NO:118; FIG. 8B), such that the ORFwas cloned under the control of the FBAIN promoter and the PEX20-3′terminator region. However, since the DGAT1 ORF contained an internalNcoI site, it was necessary to perform two separate restriction enzymedigestions for cloning. First, ˜2 μg of the purified PCR product wasdigested with BamHI and Nco I. The reaction mixture contained 20 U ofeach enzyme (Promega) and 6 μl of restriction buffer D in a total volumeof 60 μl. The mixture was incubated for 2 hrs at 37° C. A ˜320 bpfragment was separated by agarose gel electrophoresis and purified usinga Qiagen Qiaex II gel purification kit. Separately, ˜2 μg of thepurified PCR product was digested with BamHI and Not I using identicalreaction conditions to those above, except Nco I was replaced by Not I.A ˜1280 bp fragment was isolated and purified as above. Finally, ˜3 μgof pZUF17 was digested with Nco I and Not I and purified as describedabove, generating a ˜7 kB fragment.

The ˜7 kB Nco I/Not I pZUF17 fragment, the ˜320 bp Nco I/BamHI DGAT1fragment and the ˜1280 bp BamHI/Not I DGAT1 fragment were ligatedtogether in a three-way ligation incubated at room temperatureovernight. The ligation mixture contained 100 ng of the 7 kB fragmentand 200 ng each of the 320 bp and 1280 bp fragments, 2 μl ligase buffer,and 2 U T4 DNA ligase (Promega) in a total volume of 20 μl. The ligationproducts were used to transform E. coli Top10 chemical competent cells(Invitrogen) according to the manufacturer's protocol.

Individual colonies (12 total) from the transformation were used toinoculate cultures for miniprep analysis. Restriction mapping andsequencing showed that 5 out of the 12 colonies harbored the desiredplasmid, which was named “pMDGAT1-17” (FIG. 15C; SEQ ID NO:141).

Y. lipolytica strain Y2067U (from Example 10) was transformed withpMDGAT1-17 and pZUF-MOD-1 (supra, Example 14), respectively, accordingto the General Methods. Transformants were grown for 2 days in syntheticMM supplemented with amino acids, followed by 4 days in HGM. The fattyacid profile of two transformants containing pMDGAT1-17 and twotransformants containing pZUF-MOD-1 are shown below in Table 35, basedon GC analysis (as described in the General Methods). Fatty acids areidentified as 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETAand EPA; and the composition of each is presented as a % of the totalfatty acids.

TABLE 35 Lipid Composition In Yarrowia Strain Y2067U Engineered ToOverexpress M. alpina DGAT1 Total Fatty Acids Strain 18:0 18:1 18:2 GLADGLA ARA ETA EPA Y2067U + pZUF-MOD-1 #1 1.31 6.92 12.03 23.11 5.72 1.053.80 13.20 Y2067U + pZUF-MOD-1 #2 1.39 6.83 12.15 21.99 5.83 1.07 3.8213.47 Y2067U + pMDGAT1-17 #1 0.89 7.13 10.87 24.88 5.82 1.19 3.97 14.09Y2067U + pMDGAT1-17 #2 0.86 7.20 10.25 22.42 6.35 1.26 4.38 15.07

As demonstrated above, expression of the M. alpina DGAT1 from plasmidpMDGAT1-17 increased the % EPA from ˜13.3% in the “control” strains to˜14.1% (“Y2067U+pMDGAT1-17 #1”) and ˜15.1% (“Y2067U+pMDGAT1-17 #2”),respectively. An additional increase in EPA would be expected, if thenative Yarrowia lipolytica DGAT1 (SEQ ID NOs:81 and 82) were knocked-outin e.g., strain Y2067U+pMDGAT1-17.

Example 16 Mortierella alpina DGAT2 Increases Percent PUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2067U (Example 10) that wastransformed to co-express the M. alpina DGAT2 cDNA (SEQ ID NO:95). It iscontemplated art that a Y. lipolytica host strain engineered to produceARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9elongase/Δ8 desaturase pathway could demonstrate increased ARAbiosynthesis and accumulation, if the M. alpina DGAT2 was similarlyco-expressed therein (e.g., in strains Y2034, Y2047 and/or Y2214).

The M. alpina DGAT2 ORF was cloned into plasmid pZUF17 as follows.First, the ORF was PCR-amplified using primers MDGAT-F and MDGAT-R1 (SEQID NOs:297 and 298) from the M. alpina cDNA (supra, Example 13). Theexpected 1015 bp fragment was isolated, purified, digested with Nco Iand Not I and cloned into Nco I-Not I cut pZUF17 vector (SEQ ID NO:118;FIG. 8B), such that the gene was under the control of the Y. lipolyticaFBAIN promoter and the PEX20-3′ terminator region. Correct transformantswere confirmed by restriction analysis of miniprep DNA and the resultantplasmid was designated as “pMDGAT2-17” (SEQ ID NO:142).

Y. lipolytica strain Y2067U (from Example 10) was transformed withpMDGAT2-17 and pZUF-MOD-1 (supra, Example 14), respectively, accordingto the General Methods. Transformants were grown for 2 days in syntheticMM supplemented with amino acids, followed by 4 days in HGM. The fattyacid profile of two transformants containing pMDGAT2-17 and twotransformants containing pZUF-MOD-1 are shown below based on GC analysis(as described in the General Methods). Fatty acids are identified as18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and thecomposition of each is presented as a % of the total fatty acids.

TABLE 36 Lipid Composition In Yarrowia strain Y2067U Engineered ToOverexpress M. alpina DGAT2 Total Fatty Acids Strain 18:0 18:1 18:2 GLADGLA ARA ETA EPA Y2067U + pZUF-MOD-1 #1 1.31 6.92 12.03 23.11 5.72 1.053.80 13.20 Y2067U + pZUF-MOD-1 #2 1.39 6.83 12.15 21.99 5.83 1.07 3.8213.47 Y2067U + pMDGAT2-17 #1 0.00 7.47 10.77 25.30 5.70 1.43 3.45 15.12Y2067U + pMDGAT2-17 #2 1.45 7.79 9.96 25.16 6.06 1.25 3.99 15.37

Expression of the M. alpina DGAT2 from plasmid pMDGAT2-17 increased the% EPA from ˜13.3% in the “control” strains to ˜15.25%(“Y2067U+pMDGAT2-17”). An additional increase in EPA would be expected,if the native Yarrowia lipolytica DGAT2 (SEQ ID NOs:89-94) wereknocked-out in e.g., strain Y2067U+pMDGAT2-17.

Example 17 Mortierella alpina GPAT Increases Percent PUFAs

The present Example describes increased DGLA biosynthesis andaccumulation (and reduced quantities of 18:1) in Yarrowia lipolyticastrain Y2107U1 (Example 11) that was transformed to co-express the M.alpina GPAT ORF (SEQ ID NO:97). It is contemplated that a Y. lipolyticahost strain engineered to produce ARA via either the Δ6 desaturase/Δ6elongase pathway or the Δ9 elongase/Δ8 desaturase pathway coulddemonstrate increased ARA biosynthesis and accumulation, if the M.alpina GPAT was similarly co-expressed therein (e.g., in strains Y2034,Y2047 and/or Y2214).

Identification of a M. Alpina GPAT Using Degenerate PCR Primers

Based on sequences of GPAT from Aspergillus nidulans (GenBank AccessionNo. EAA62242) and Neurospora crassa (GenBank Accession No.XP_(—)325840), the following primers were designed for degenerate PCR:

MGPAT-N1 (SEQ ID NO: 299) CCNCAYGCNAAYCARTTYGT MGPAT-NR5(SEQ ID NO: 300) TTCCANGTNGCCATNTCRTC[Note: The nucleic acid degeneracy code usedfor SEQ ID NOs: 299 and 300 was as follows: R = S/G; Y = C/T; and N =A/C/T/G.]

PCR amplification was carried out in a Perkin Elmer GeneAmp 9600 PCRmachine using TaKaRa ExTaq premix Taq polymerase (TaKaRa Bio Inc., Otsu,Shiga, Japan). Amplification was carried out as follows: 30 cycles ofdenaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec andelongation at 72° C. for 90 sec, followed by a final elongation cycle at72° C. for 7 min.

A fragment of ˜1.2 kB was obtained (SEQ ID NO:99). This fragment waspurified with a Qiagen QiaQuick PCR purification kit, cloned into theTOPO® cloning vector pCR2.1-TOPO (Invitrogen), and sequenced. Theresultant sequence, when translated, had homology to known GPATs, basedon BLAST program analysis.

Based on the sequence of the 1212 bp cDNA fragment, the 5′ and 3′ endregions of the M. alpina GPAT were cloned by PCR amplification andgenome walking techniques. This enabled assembly of a contig,corresponding to the −1050 bp to +2885 bp region of the M. alpina GPAT(SEQ ID NO:100). This contig included the entire coding region of GPATand four introns (SEQ ID NOs:104, 105, 106 and 107).

Specifically, the M. alpina cDNA sample described in Example 13 (1 μl)was used as a template for amplification of the 3′-end of the GPAT.MGPAT-5N1 (SEQ ID NO:301) and CDSIII/3′ (SEQ ID NO:282) were used asprimers. PCR amplification was carried out in a Perkin Elmer GeneAmp9600 PCR machine using TaKaRa ExTaq premix Taq polymerase (TaKaRa BioInc., Otsu, Shiga, Japan). Amplification was carried out as follows: 30cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30sec and elongation at 72° C. for 120 sec, followed by a final elongationcycle at 72° C. for 7 min.

The PCR product was diluted 1:10, and 1 μl of diluted PCR product wasused as template for the second round of amplification, using MGPAT-5N2(SEQ ID NO:302) and CDSIII/3′ as primers. The conditions were exactlythe same as described above. The second round PCR product was againdiluted 1:10 and 1 μl of the diluted PCR product used as template for athird round of PCR, using MGPAT-5N3 (SEQ ID NO:303) and CDSIII/3′ asprimers. The PCR conditions were again the same.

A ˜1 kB fragment was generated in the third round of PCR. This fragmentwas purified with a Qiagen PCR purification kit and cloned intopCR2.1-TOPO vector for sequence analysis. Results from sequence analysisshowed that this 965 bp fragment (SEQ ID NO:101) corresponded with the3′-end of the GPAT gene.

A Clontech Universal GenomeWalker™ kit was used to obtain a piece ofgenomic DNA corresponding to the 5′-end region of the M. alpina GPAT.Briefly, 2.5 μg each of M. alpina genomic DNA was digested with DraI,EcoRV, PvuII or StuI individually, the digested DNA samples werepurified using Qiagen Qiaquick PCR purification kits and eluted with 30μl each of kit buffer EB, and the purified samples were then ligatedwith Genome Walker adaptor (SEQ ID NOs:304 [top strand] and 305 [bottomstrand]), as shown below:

5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGG T-3′3′-H2N-CCCGACCA-5′Each ligation reaction mixture contained 1.9 μl of 25 μM Genome Walkeradaptor, 1.6 μl 10× ligation buffer, 0.5 μl T4 DNA ligase and 4 μl ofone of the purified digested genomic DNA samples. The reaction mixtureswere incubated at 16° C. overnight. The reaction was terminated byincubation at 70° C. for 5 min. Then, 72 μl of 10 mM TrisHCl, 1 mM EDTA,pH 7.4 buffer was added to each ligation reaction mix.

Four separate PCR reactions were performed, each using one of the fourligation mixtures as template. The PCR reaction mixtures contained 1 μlof ligation mixture, 0.5 μl of 20 μM MGPAT-5-1A (SEQ ID NO:306), 1 μl of10 μM kit primer AP1 (SEQ ID NO:307), 22.5 μl water, and 25 μl ExTaqpremix Taq 2×PCR solution (TaKaRa). The PCR reactions were carried outfor 32 cycles using the following conditions: denaturation at 94° C. for30 sec, annealing at 55° C. for 30 sec, and elongation at 72° C. for 180sec. A final elongation cycle at 72° C. for 7 min was carried out,followed by reaction termination at 4° C.

The products of each PCR reaction were diluted 1:50 individually andused as templates for a second round of PCR. Each reaction mixturecontained 1 μl of one of the diluted PCR product as template, 0.5 μl of20 μM MGPAT-3N1 (SEQ ID NO:308), 21 μl of 10 μM kit primer AP2 (SEQ IDNO:309), 22.5 μl water and 25 μl of ExTaq premix Taq 2×PCR solution(TaKaRa). PCR reactions were carried out for 32 cycles using the samethermocycler conditions described above.

A DNA fragment was obtained from the second round of PCR. This fragmentwas purified and cloned into pCR2.1-TOPO and sequenced. Sequenceanalysis showed that the 1908 bp fragment (SEQ ID NO:102) was the 5′-endof the M. alpina GPAT gene.

Similarly, a 966 bp fragment (SEQ ID NO:103) was obtained by two roundsof genome walking as described above, except using primer MGPAT-5N1 asthe gene specific primer for the first round of PCR and primer MGPAT-5N2as the gene specific primer for the second round. This fragment was alsopurified, cloned into pCR2.1-TOPO and sequenced. Sequence analysisshowed that it contained a portion of the GPAT gene; however, thefragment was not long enough to extend to either end of the gene.Comparison with the 3′ cDNA sequence (SEQ ID NO:101) showed that thelast 171 bp of the ORF was not included.

Assembly of the Full-Length GPAT Sequence from Mortierella alpina

A 3935 bp sequence (SEQ ID NO:100) containing the complete GPAT gene(comprising a region extending 1050 bases upstream of the GPATtranslation initiation ‘ATG’ codon and extending 22 bases beyond theGPAT termination codon) was assembled from the sequences of the originalpartial cDNA fragment (SEQ ID NO:99), the 3′ cDNA fragment (SEQ IDNO:101), the internal genomic fragment (SEQ ID NO:103), and the 5′genomic fragment (SEQ ID NO:102) described above (graphicallyillustrated in FIG. 16). Included in this region is the 2151 bp GPATORF. The complete nucleotide sequence of the M. alpina GPAT ORF from‘ATG’ to the stop codon ‘TAG’ is provided as SEQ ID NO:97 (correspondingto bases 1050 to 2863 of SEQ ID NO:100, excluding the four introns(i.e., intron 1 [SEQ ID NO:104], corresponding to bases 1195 to 1469 ofSEQ ID NO:100; intron 2 [SEQ ID NO:105], corresponding to bases 1585 to1839 of SEQ ID NO:100; intron 3 [SEQ ID NO:106], corresponding to bases2795 to 2877 of SEQ ID NO:100 and intron 4 [SEQ ID NO:107],corresponding to bases 2940 to 3038 of SEQ ID NO:100). The translatedamino acid sequence (SEQ ID NO:98) showed homology with a number offungal, plant and animal GPATs.

More specifically, identity of the sequence was determined by conductingBLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J.Mol. Biol. 215:403-410 (1993)) searches. The amino acid fragmentdescribed herein as SEQ ID NO:98 had 47% identity and 65% similaritywith the protein sequence of the putative GPAT of Ustilago maydis(GenBank Accession No. EAK84237), with an expectation value of 1e-152;additionally, SEQ ID NO:98 had 47% identity and 62% similarity with theGPAT of Aspergillus fumigatus (GenBank Accession No. EAL20089), with anexpectation value of 1e-142.

Construction of Plasmid pMGPAT-17, Comprising a FBAIN::MGPAT::PEX20-3′Chimeric Gene

The M. alpina GPAT ORF was cloned as follows. Primers mgpat-cdna-5 andmgpat-cdna-R (SEQ ID NOs:310 and 311) were used to amplify the GPAT ORFfrom the cDNA of M. alpina bp PCR. The reaction mixture contained 1 μlof the cDNA, 1 μl each of the primers, 22 μl water and 25 μl ExTaqpremix 2×Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga, 520-2193,Japan). Amplification was carried out as follows: initial denaturationat 94° C. for 150 sec, followed by 30 cycles of denaturation at 94° C.for 30 sec, annealing at 55° C. for 30 sec and elongation at 72° C. for120 sec. A final elongation cycle at 72° C. for 10 min was carried out,followed by reaction termination at 4° C. An ˜2.2 kB DNA fragment wasobtained from the PCR reaction. It was purified using a Qiagen PCRpurification kit according to the manufacturer's protocol.

The purified PCR product was digested with BamHI and EcoRI, and a ˜470bp fragment was isolated by gel agarose electrophoresis and purifiedusing a Qiagen gel purification kit. Separately, the PCR product wasalso cut with EcoRI and NotI, and a 1.69 kB fragment isolated andpurified as above. The two fragments were ligated into BamHI and NotIcut pZUF-MOD-1 vector (SEQ ID NO:140; FIG. 15B), such that the gene wasunder the control of the Y. lipolytica FBAIN promoter and the PEX20-3′terminator region in the auto-replicating vector for expression in Y.lipolytica. Correct transformants were confirmed by restriction analysisof miniprep DNA and the resultant plasmid was designated as “pMGPAT-17”(SEQ ID NO:143; FIG. 15D).

Analysis of Lipid Composition in Transformant Y. lipolyticaOver-Expressing M. alpina GPAT

Y. lipolytica strain Y2107U1 (from Example 11) was transformed withplasmid pMGPAT-17 and plasmid pZUF-MOD-1 (supra, Example 14),respectively, according to the General Methods. Transformants were grownfor 2 days in synthetic MM supplemented with amino acids, followed by 4days in HGM. The fatty acid profile of two transformants containingpZUF-MOD-1 and four transformants containing pMGPAT-17, are shown belowin the Table, based on GC analysis (as described in the GeneralMethods). Fatty acids are identified as 18:0, 18:1 (oleic acid), 18:2(LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each ispresented as a % of the total fatty acids.

TABLE 37 Lipid Composition In Yarrowia Strain Y2107U1 Engineered ToOver- Express M. alpina GPAT Total Fatty Acids Strain 18:0 18:1 18:2 GLADGLA ARA ETA EPA Y2107U1 + pZUF-MOD-1 #1 2.8 22.7 9.8 28.5 2.7 1.7 0.417.4 Y2107U1 + pZUF-MOD-1 #2 2.5 23.4 10.3 28.7 2.5 1.5 0.3 16.8Y2107U1 + pMGPAT-17 #1 3.2 14.8 11.7 29.8 5.6 2.0 0.3 18.4 Y2107U1 +pMGPAT-17 #2 2.9 16.3 11.7 28.3 6.1 1.8 0.4 16.9 Y2107U1 + pMGPAT-17 #32.1 14.3 10.8 27.5 7.2 1.4 0.4 17.4 Y2107U1 + pMGPAT-17 #4 2.7 15.7 11.529.1 6.3 1.7 0.4 17.3

As demonstrated above, expression of the M. alpina GPAT from pMGPAT-17increased the % DGLA from ˜2.5% in the “control” strains to 6.5%. Thelevel of 18:1 decreased from ˜23% to ˜16%. An additional increase inDGLA (or any other downstream PUFAs) would be expected, if the nativeYarrowia lipolytica GPAT was knocked-out in a transformant strainexpressing pMGPAT-17.

Example 18 Mortierella alpina Fatty Acid Elongase “ELO3” IncreasesPercent PUFAs Content

The present Example describes 35% more C18 fatty acids (18:0, 18:1, 18:2and GLA) and 31% less C16 fatty acids in Yarrowia lipolytica strainY2031 (Example 5) that was transformed to co-express the M. alpinaC_(16/18) fatty acid elongase (“ELO3”; SEQ ID NOs:53 and 54), relativeto control strains. It is contemplated that ELO3 (which could optionallybe codon-optimized for increased expression), could push carbon fluxinto either the engineered Δ6 desaturase/Δ6 elongase pathway or the Δ9elongase/Δ8 desaturase pathway as a means to increase production of thedesired PUFA, i.e., ARA. For example, a chimeric gene comprising thisC_(16/18) fatty acid elongase could readily be introduced into e.g.,strains Y2034, Y2047 or Y2214.

Sequence Identification of a M. Alpina C_(16/18) Fatty Acid Elongase

A cDNA fragment (SEQ ID NO:55) encoding a portion of a M. alpina fattyacid elongase was identified from among 9,984 M. alpina cDNA sequences(Example 13). This cDNA fragment bore significant homology to a numberof fatty acid elongases and thus was tentatively identified as anelongase.

The results of the BLAST comparison summarizing the sequence to whichSEQ ID NO:55 had the most similarity are reported according to the %identity, % similarity, and Expectation value. Specifically, thetranslated amino acid sequence of SEQ ID NO:55 had 32% identity and 46%similarity with the protein sequence of a potential fatty acid elongasefrom Candida albicans SC5314 (GenBank Accession No. EAL04510.1,annotated therein as one of three potential fatty acid elongase genessimilar to S. cerevisiae EUR4, FEN1 and ELO1), with an expectation valueof 4e-13. Additionally, SEQ ID NO:55 had 35% identity and 53% similaritywith ELO1 from Saccharomyces cerevisiae (GenBank Accession No.NC_(—)001142, bases 67849-68781 of chromosome X). The S. cerevisiae ELO1is described as a medium-chain acyl elongase, that catalyzescarboxy-terminal elongation of unsaturated C12-C16 fatty acyl-CoAs toC16-018 fatty acids.

On the basis of the homologies reported above, the Yarrowia lipolyticagene product of SEQ ID NO:55 was designated herein as “elongase 3” or“ELO3”.

Analysis of the partial fatty acid elongase cDNA sequence (SEQ ID NO:55)indicated that the 5′ and 3′-ends were both incomplete. To obtain themissing 3′ region of the M. alpina ELO3, a Clontech UniversalGenomeWalker™ kit was used (as described in Example 17). Specifically,the same set of four ligation mixtures were used for a first round ofPCR, using the same components and conditions as described previously,with the exception that MA Elong 3′1 (SEQ ID NO:312) and AP1 were usedas primers (i.e., instead of primers MGPAT-5-1A and AP1). The secondround of PCR used MA Elong 3′2 (SEQ ID NO:313) and AP2 as primers. A1042 bp DNA fragment was obtained from the second round of PCR (SEQ IDNO:56). This fragment was purified and cloned into pCR2.1-TOPO andsequenced. Sequence analysis showed that the fragment contained the3′-end of ELO3, including ˜640 bp downstream of the ‘TAA’ stop codon ofthe gene.

The same set of four ligation mixtures used in the Clontech 3′-end RACE(supra) were also used to obtain the 5′-end region of the M. alpinaELO3. Specifically, a first round of PCR using the same components andconditions as described above was conducted, with the exception that MAElong 5′1 (SEQ ID NO:314, nested at the 5′ end) and AP1 were used asprimers (i.e., instead of primers MA Elong 3′1 and AP1). The secondround of PCR used MA Elong 5′2 (SEQ ID NO:315, nested at the 5′ end) andAP2 as primers. A 2223 bp DNA fragment (SEQ ID NO:57) was obtained. Itwas purified and cloned into pCR2.1-TOPO and sequenced. Analysis of thesequence showed that it contained the 5′-region of the ELO3 gene.

Thus, the entire cDNA sequence of the M. alpina ELO3 (SEQ ID NO:53) wasobtained by combining the original partial cDNA sequence (SEQ ID NO:55)with the overlapping 5′ and 3′ sequences obtained by genome walking (SEQID NOs:57 and 56, respectively; graphically illustrated in FIG. 17).This yielded a sequence of 3557 bp, identified herein as SEQ ID NO:58,comprising: 2091 bp upstream of the putative ‘ATG’ translationinitiation codon of ELO3; the 828 bp ELO3 ORF (i.e., SEQ ID NO:53,corresponding to bases 2092-2919 of SEQ ID NO:58); and, 638 bpdownstream of the ELO3 stop codon (corresponding to bases 2920-3557 ofSEQ ID NO:58).

The corresponding genomic sequence of the M. alpina ELO3 is longer thanthe cDNA fragment provided as SEQ ID NO:58. Specifically, a 542 bpintron (SEQ ID NO:59) was found in the genomic DNA containing the ELO3gene at 318 bp of the ORF; thus, the genomic DNA fragment, providedherein as SEQ ID NO:60, is 4,099 bp (FIG. 17).

The translated ELO3 protein sequence (SEQ ID NO:54) had the followinghomology, based on BLAST program analysis: 37% identity and 51%similarity to the potential fatty acid elongase from Candida albicansSC5314 (GenBank Accession No. EAL04510.1), with an expectation value of4e-43. Additionally, the translated ELO3 shared 33% identity and 44%similarity with the protein sequence of XP_(—)331368 (annotated thereinas a “hypothetical protein”) from Neurospora crassa, with an expectationvalue of 3e-44.

Construction of Plasmid pZUF6S-E3WT, Comprising a FBAIN::ELO3::PEX16-3′Chimeric Gene

The M. alpina fatty acid ELO3 ORF was cloned as follows. Primers MAElong 5′ NcoI 3 and MA Elong 3′ NotI (SEQ ID NOs:316 and 317) were usedto amplify the ELO3 ORF from the cDNA of M. alpina (Example 13) by PCR.The reaction mixture contained 1 μl of the cDNA, 1 μl each of theprimers, 22 μl water and 25 μl ExTaq premix 2×Taq PCR solution (TaKaRa).Amplification was carried out as follows: initial denaturation at 94° C.for 30 sec, followed by 32 cycles of denaturation at 94° C. for 30 sec,annealing at 55° C. for 30 sec and elongation at 72° C. for 120 sec. Afinal elongation cycle at 72° C. for 7 min was carried out, followed byreaction termination at 4° C. An ˜830 bp DNA fragment was obtained fromthe PCR reaction. It was purified using a Qiagen (Valencia, Calif.) PCRpurification kit according to the manufacturer's protocol. The purifiedPCR product was divided into two aliquots, wherein one was digested withNcoI and NspI, while the other with NspI and NotI. The ˜270 bp NcoI-NspIand ˜560 bp NspI-NotI fragments were cloned into Nco I-Not I cutpZF5T-PPC vector (FIG. 11C; SEQ ID NO:122) by three-piece ligation, suchthat the gene was under the control of the Y. lipolytica FBAIN promoterand the PEX16-3′ terminator region (GenBank Accession No. U75433) in theauto-replicating vector for expression in Y. lipolytica. Correcttransformants were confirmed by miniprep analysis and the resultantplasmid was designated as “pZF5T-PPC-E3” (SEQ ID NO:144).

Plasmid pZF5T-PPC-E3 was digested with ClaI and PacI and the ˜2.2 kBband (i.e., the FBAIN::ELO 3::PEX16-3′ fragment) was purified from anagarose gel using a Qiagen gel extraction kit. The fragment was clonedinto ClaI-PacI cut pZUF6S (FIG. 18A; SEQ ID NO:145), an auto-replicationplasmid containing the Mortierella alpina Δ6 desaturase ORF (“D65”;GenBank Accession No. AF465281) under the control of the FBAIN promoterwith a Pex20-3′ terminator (i.e., a FBAIN::D6S::Pex20 chimeric gene) anda Ura3 gene. Correct transformants were confirmed by miniprep analysisand the resultant plasmid was designated as “pZUF6S-E3WT” (FIG. 18B; SEQID NO:146).

Analysis of Lipid Composition in Transformant Y. LipolyticaOver-Expressing the M. Alpina ELO3

Y. lipolytica strain Y2031 (Example 5) was transformed with plasmidpZUF6S (control, comprising a FBAIN::D6S::Pex20 chimeric gene) andplasmid pZUF6S-E3WT (comprising a FBAIN::D6S::Pex20 chimeric gene andthe FBAIN::ELO 3::PEX16 chimeric gene) according to the General Methods.Transformants were grown for 2 days in synthetic MM supplemented withamino acids, followed by 4 days in HGM. The fatty acid profile of sixclones containing pZUF6S (clones #1-6, from a single transformation) and22 clones (from four different transformations [i.e., #3, 5, 6, and 7])containing pZUF6S-E3WT are shown below in Table 38, based on GC analysis(as described in the General Methods). Fatty acids are identified as16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2(LA) and GLA; and the composition of each is presented as a % of thetotal fatty acids.

TABLE 38 Lipid Composition In Yarrowia Strain Y2031 Engineered ToOver-Express M. alpina ELO3

 Strain Y2031 Transformant Fatty Acid Composition (% Of Total FattyAcids) And/Or Clone No. 16:0 16:1 18:0 18:1 18:2 GLA pZUF6S #1 (control)9.0 23.2 1.2 38.2 19.8  6.9 pZUF6S #2 (control) 10.1  23.4 1.4 39.0 17.5 7.1 pZUF6S #3 (control) 9.7 22.7 1.4 39.0 20.2  7.0 pZUF6S #4 (control)8.5 24.1 0.0 40.8 19.8  6.9 pZUF6S #5 (control) 9.8 22.4 1.7 39.1 20.2 6.8 pZUF6S #6 (control) 9.1 22.7 1.9 39.9 19.7  6.6 pZUF6S-E3WT #3-18.9 17.3 4.1 36.5 21.6 11.6 pZUF6S-E3WT #3-2 8.8 17.8 3.7 36.9 21.3 11.5

pZUF6S-E3WT #3-6 8.5 19.9 4.4 37.8 17.1 12.3 pZUF6S-E3WT #5-1 8.6 17.64.0 37.6 21.1 11.1 pZUF6S-E3WT #5-2 8.8 17.1 3.9 37.6 21.3 11.2pZUF6S-E3WT #5-3 9.1 17.1 3.5 37.6 21.5 11.1 pZUF6S-E3WT #5-4 8.8 17.94.3 38.0 19.3 11.7 pZUF6S-E3WT #5-5 9.2 16.1 4.4 37.0 21.6 11.7

pZUF6S-E3WT #6-1 9.4 16.9 4.6 36.6 21.5 11.0 pZUF6S-E3WT #6-2 9.8 16.24.1 36.5 21.9 11.6 pZUF6S-E3WT #6-3 9.4 17.0 4.4 36.2 21.8 11.3pZUF6S-E3WT #6-4 8.3 16.6 4.2 36.9 21.9 12.2 pZUF6S-E3WT #6-5 8.8 18.55.5 36.0 17.8 13.4 pZUF6S-E3WT #6-6 8.7 19.5 5.2 35.4 18.1 13.2

pZUF6S-E3WT #7-2 8.0 17.7 4.0 37.7 20.9 11.7

pZUF6S-E3WT #7-5 8.3 17.0 4.7 36.7 21.2 12.1 pZUF6S-E3WT #7-6 8.0 18.04.8 36.3 20.8 12.1

Some of the samples (labeled in bold and italics) deviated from expectedreadings. Specifically, neither Y2031+pZUF6S-E3WT #3-3 norY2031+pZUF6S-E3WT #5-6 produced GLA. Similarly, Y2031+pZUF6S-E3WT #7-1,#7-3 and #7-4 had GC errors, wherein the 16:0 and 16:1 peaks were readby the GC as a single peak. As a result of these variant results, Table39 reports the average lipid in the control and transformant strainsexpressing ELO3. Specifically, Table 39 shows the averages from thefatty acid profiles in Table 38, although the lines indicated by boldand italics as being incorrect in Table 38 were not included whencalculating these averages. “Total C16” represents the sum of theaverage areas of 16:0 and 16:1, while “Total C18” reflects the sum ofthe average areas of 18:0, 18:1, 18:2 and GLA.

TABLE 39 Average Lipid Composition In Yarrowia Strain Y2031 EngineeredTo Over-Express M. alpina ELO3 Y. lipolytica Average Fatty AcidComposition Strain Y2031 (% Of Total Fatty Acids) Total TotalTransformant 16:0 16:1 18:0 18:1 18:2 GLA C16 C18 pZUF6S 9.4 23.1 1.339.3 19.5 6.9 32.4 67.1 (control) pZUF6S- 8.7 18.3 4.1 37.1 20.0 11.827.0 73.0 E3WT #3 pZUF6S- 8.9 17.2 4.0 37.6 21.0 11.4 26.1 73.9 E3WT #5pZUF6S- 9.1 17.5 4.6 36.3 20.5 12.1 26.5 73.5 E3WT #6 pZUF6S- 8.1 17.64.5 36.9 21.0 12.0 25.6 74.4 E3WT #7

Based on the data reported above, overexpression of the M. alpina ELO3resulted in an increased percentage of C18 and a reduced percentage ofC16 when co-expressed with a M. alpina Δ6 desaturase in Yarrowialipolytica strain Y2031, relative to a control strain of Y2031overexpressing the M. alpina Δ6 desaturase only. This indicated that theM. alpina ELO3 was indeed a C_(16/18) fatty acid elongase.

Example 19 Yarrowia C _(16/18) Fatty Acid Elongase “YE2” IncreasesPercent PUFAs

The present Example describes increased GLA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2031 (Example 5) that wastransformed to co-express the Y. lipolytica C _(16/18) fatty acidelongase (“YE2”; SEQ ID NO:62). It is contemplated that the YE2 elongasecould push carbon flux into either the engineered Δ6 desaturase/Δ6elongase pathway or the Δ9 elongase/Δ8 desaturase pathway as a means toincrease production of the desired PUFA, i.e., ARA. For example, achimeric gene comprising this C_(16/18) fatty acid elongase couldreadily be introduced into e.g., strains Y2034, Y2047 or Y2214.

Sequence Identification of a Yarrowia lipolytica C _(16/18) Fatty AcidElongase

A novel fatty acid elongase candidate from Y. lipolytica was identifiedby sequence comparison using the rat Elo2 C_(16/18) fatty acid elongaseprotein sequence (GenBank Accession No. AB071986; SEQ ID NO:51) as aquery sequence. Specifically, this rElo2 query sequence was used tosearch GenBank and the public Y. lipolytica protein database of the“Yeast project Genolevures” (Center for Bioinformatics, LaBR1, TalenceCedex, France) (see also Dujon, B. et al., Nature 430 (6995):35-44(2004)). This resulted in the identification of a homologous sequence,GenBank Accession No. CAG77901 (SEQ ID NOs:61 and 62), annotated as an“unnamed protein product”). This gene was designated as YE2.

Comparison of the Yarrowia YE2 amino acid sequences to public databases,using a BLAST algorithm (Altschul, S. F., et al., Nucleic Acids Res.25:3389-3402 (1997)), revealed that the most similar known amino acidsequence was that from Candida albicans SC5314 (SEQ ID NO:63, GenBankAccession No. EAL04510), annotated as a probable fatty acid elongase.The proteins shared about 40% identity and scored at 236 with an E valueof 7e-61.

Isolation of Yarrowia YE2 Gene

The coding region of the YE2 gene was amplified by PCR using Yarrowiagenomic DNA as template and oligonucleotides YL597 and YL598 (SEQ IDNOs:318 and 319) as primers. The PCR reaction was carried out in a 50 μltotal volume, as described in the General Methods. The thermocyclerconditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30sec, 72° C. for 1 min, followed by a final extension at 72° C. for 10min. The PCR products of the YE2 coding region were purified, digestedwith NcoI/NotI, and then ligated with NcoI/NotI digested pZKUGPYE1-N(infra, Example 20; see also FIG. 18C, SEQ ID NO:147) to generatepZKUGPYE2 (FIG. 18D, SEQ ID NO:148). The addition of a NcoI site aroundthe ‘ATG’ translation initiation codon changed the second amino acid ofYE2 from L to V.

The ClaI/NotI fragment of pZKUGPYE2 (containing the GPAT promoter andYE2 coding region) and a NotI/PacI fragment containing the Acoterminator (prepared by PCR amplifying the ACO 3′ terminator withprimers YL325 and YL326 [SEQ ID NOs:371 and 372] and then digesting withNotI/PacI), were directionally ligated with ClaI/PacI digested vectorpZUF6S to produce pZUF6YE2. The ClaI/NcoI fragment of pZKUT16(containing the TEF promoter) and the NcoI/PacI fragment of pZUF6YE2(containing the coding region of YE2 and the Aco terminator) weresubsequently directionally ligated with ClaI/PacI digested vector pZUF6Sto produce pZUF6TYE2 (SEQ ID NO:149).

Analysis of Lipid Composition in Transformant Y. lipolyticaOver-Expressing YE2

Plasmid pZUF6S (FIG. 18A, SEQ ID NO:145) and pZUF6TYE2 (SEQ ID NO:149)were used to separately transform Yarrowia strain Y2031. The componentsof these two plasmids are described in Tables 40 and 41.

TABLE 40 Description of Plasmid pZUF6S (SEQ ID NO: 145) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:145 Components EcoRI/ClaI Yarrowia autonomous replicating sequence 18(3114-4510) (ARS18; GenBank Accession No. M91600) SalI/PacI YarrowiaUra3 gene (GenBank Accession No. (6022-4530) AJ306421) EcoRI/BsiWIFBAIN::Δ6S::Pex20, comprising: (6063-318) FBAIN: FBAIN promoter (SEQ IDNO: 162) Δ6S: codon-optimized Δ6 desaturase gene (SEQ ID NO: 3), derivedfrom Mortierella alpina (GenBank Accession No. AF465281) Pex20: Pex20terminator sequence from Yarrowia Pex20 gene (GenBank Accession No.AF054613)

TABLE 41 Description of Plasmid pZUF6TYE2 (SEQ ID NO: 149) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:149 Components EcoRI/ClaI Yarrowia autonomous replicating sequence 18(7461-8857) (ARS18; GenBank Accession No. M91600) SalI/PacI YarrowiaUra3 gene (GenBank Accession No. (1907-415) AJ306421) EcoRI/BsiWIFBAIN::Δ6S::Pex20: as described for pZUF6 (1948-4665) (supra) ClaI/PacITEF::YE2::Aco, comprising: (8857-415) TEF: TEF promoter (GenBankAccession No. AF054508) YE2: coding region of Yarrowia YE2 gene (SEQ IDNO: 61; GenBank Accession No. CAG77901) Aco: Aco3 terminator sequence ofYarrowia Aco3 gene (GenBank Accession No. AJ001301)

Y. lipolytica strain Y2031 (Example 5) was transformed with plasmidpZUF6S (control) and plasmid pZUF6TYE2 according to the General Methods.Transformants were grown for 2 days in liquid MM. The fatty acid profileof eight colonies each containing pZUF6S or pZUF6YE2 are shown below inTable 42, based on GC analysis (as described in the General Methods).Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0, 18:1 (oleic acid), 18:2 (LA) and GLA; and the composition of eachis presented as a % of the total fatty acids.

TABLE 42 Comparison Of Fatty Acid Composition In Yarrowia Strain Y2031Transformed With pZUF6S And pZUF6TYE2 Y. lipolytica Strain Y2031 FattyAcid Composition (% Of Total Fatty Acids) Transformants 16:0 16:1 18:018:1 18:2 GLA pZUF6S #1 (control) 15.4 13.8 2.5 34.1 16.8 8.3 pZUF6S #2(control) 15.2 12.8 3.0 36.5 16.4 8.3 pZUF6S #3 (control) 15.1 12.2 3.236.5 17.1 8.5 pZUF6S #4 (control) 15.2 12.8 3.1 36.3 16.6 8.4 pZUF6S #5(control) 14.9 10.9 3.6 37.4 18.0 8.7 pZUF6S #6 (control) 14.8 10.1 4.237.6 18.7 8.6 pZUF6S #7 (control) 14.7 11.9 3.0 36.0 17.8 9.1 pZUF6S #8(control) 14.9 12.6 2.9 35.9 17.3 8.8 Average 15.0 12.1 3.2 36.3 17.38.6 pZUF6TYE2 #1 13.1 8.4 4.4 42.4 16.8 9.7 pZUF6TYE2 #2 13.1 7.6 5.340.8 18.6 9.8 pZUF6TYE2 #3 13.5 8.1 4.6 39.2 19.0 10.6 pZUF6TYE2 #4 13.47.4 5.7 39.9 18.7 9.8 pZUF6TYE2 #5 13.4 8.4 5.5 45.2 14.3 7.6 pZUF6TYE2#6 13.4 7.4 5.5 39.3 19.2 10.5 pZUF6TYE2 #7 13.4 8.6 4.4 40.6 17.9 9.9pZUF6TYE2 #8 13.2 7.5 5.4 41.2 18.0 9.7 Average 13.3 8.0 5.0 41.1 17.89.7

GC analyses showed that there were about 27.1% C16 (C16:0 and C16:1) and62.2% C18 (C18:0, C18:1, C18:2 and GLA) of total lipids produced in theY2031 transformants with pZUF6S; there were about 21.3% C16 and 73.6%C18 produced in the Y2031 transformants with pZUF6TYE2. Thus, the totalamount of C16 was reduced about 21.4%, and the total amount of C18 wasincreased about 18% in the pZUF6TYE2 transformants (as compared with thetransformants with pZUF6S). These data demonstrated that YE2 functionsas a C_(16/18) fatty acid elongase to produce C18 fatty acids inYarrowia. Additionally, there was about 12.8% more GLA produced in thepZUF6TYE2 transformants relative to the GLA produced in pZUF6Stransformants. These data suggested that the YE2 elongase could pushcarbon flux into the engineered PUFA pathway to produce more finalproduct (i.e., GLA).

Example 20 Yarrowia C_(14/16) Fatty Acid Elongase “YE1” IncreasesPercent PUFAs

The present Example describes increased GLA biosynthesis andaccumulation in Y. lipolytica strain Y2031 (Example 5) that wastransformed to co-express the Y. lipolytica C_(14/16) fatty acidelongase (“YE1”; SEQ ID NO:65). It is contemplated that the YE1 elongasecould push carbon flux into either the engineered Δ6 desaturase/Δ6elongase pathway or the Δ9 elongase/Δ8 desaturase pathway as a means toincrease production of the desired PUFA, i.e., ARA. Specifically, achimeric gene comprising this C_(14/16) fatty acid elongase couldreadily be introduced into strains Y2034, Y2047 or Y2214.

Sequence Identification of a Yarrowia Lipolytica C _(14/16) Fatty AcidElongase

A novel fatty acid elongase candidate from Yarrowia lipolytica wasidentified by sequence comparison using the rat Elo2 C_(16/18) fattyacid elongase protein sequence (GenBank Accession No. AB071986; SEQ IDNO:51) as a query sequence, in a manner similar to that used in Example19. This resulted in the identification of a homologous sequence,GenBank Accession No. CAG83378 (SEQ ID NOs:64 and 65), annotated as an“unnamed protein product”. This gene was designated as “YE1”.

Comparison of the Yarrowia YE1 amino acid sequences to public databases,using a BLAST algorithm (Altschul, S. F., et al., Nucleic Acids Res.25:3389-3402 (1997)), revealed that the most similar known sequence wasFEN1 from Neurospora crassa (GenBank Accession No. CAD70918; SEQ IDNO:66), a probable fatty acid elongase sharing about 60% identity toYE1.

Isolation of Yarrowia YE1 Gene

The DNA sequence of YE1 gene (SEQ ID NO:64) possesses an internal NcoIsite. In order to incorporate the Yarrowia translation motif around the‘ATG’ translation initiation codon of the YE1 gene, a two-step strategywas employed to PCR the entire YE1 gene from Yarrowia. Specifically,using Yarrowia genomic DNA as template, the first half of YE1 wasamplified by PCR using oligonucleotides YL567 and YL568 (SEQ ID NOs:320and 321) as primers, while the second half of the YE1 gene was amplifiedsimilarly using oligonucleotides YL569 and YL570 (SEQ ID NOs:322 and323) as primers. The PCR reactions were carried out in a 50 μl totalvolume, as described in the General Methods. The thermocycler conditionswere set for 35 cycles at 95° C. for 1 min, 56° C. for 30 sec, 72° C.for 1 min, followed by a final extension at 72° C. for 10 min. The PCRproducts corresponding to the 5′ portion of YE1 were purified and thendigested with NcoI and SacI to yield the YE1-1 fragment, while the PCRproducts of the 3′ portion of YE1 were purified and digested with SacIand NotI to yield the YE1-2 fragment. The YE1-1 and YE1-2 fragments weredirectly ligated with NcoI/NotI digested pZKUGPE1S (supra, Example 11)to generate pZKUGPYE1 (FIG. 19A, SEQ ID NO:150). The internal NcoI siteof YE1 was then mutated by site-directed mutagenesis using pZKUGPYE1 astemplate and oligonucleotides YL571 and YL572 (SEQ ID NOs:324 and 325)as primers to generate pZKUGPYE1-N (SEQ ID NO:147). Sequence analysisshowed that the mutation did not change the amino acid sequence of YE1.The addition of the NcoI site around the ATG translation initiationcodon changed the second amino acid of YE1 from S to A.

The ClaI/NcoI fragment of pZF5T-PPC (containing the FBAIN promoter) andthe NcoI/PacI fragment of pZKUGPYE1-N (containing the coding region ofYE1 and the Aco terminator) were directionally ligated withClaI/PacI-digested vector pZUF6S to produce pZUF6FYE1 (SEQ ID NO:151).

Analysis of Lipid Composition in Transformant Y. lipolyticaOver-Expressing YE1

A chimeric gene comprising the Y. lipolytica YE1 ORF was cloned intoplasmid pZUF6, such that the effect of the gene's overexpression couldbe determined by GC analysis of fatty acid composition in transformedYarrowia strains. Specifically, the components of control plasmid pZUF6S(FIG. 18A; SEQ ID NO:145, comprising a FBAIN::D6S::Pex20 chimeric gene)are described in Example 19, while those components of pZUF6FYE1 (FIG.19B; SEQ ID NO:151, comprising a FBAIN::D6S::Pex20 chimeric gene and theFBAIN::YE1::Aco chimeric gene) are described in Table 43 below.

TABLE 43 Description Of Plasmid pZUF6FYE1 (SEQ ID NO: 151) RE Sites AndNucleotides Within SEQ Description Of Fragment And Chimeric Gene ID NO:151 Components EcoRI/ClaI Yarrowia autonomous replicating sequence 18(7047-8445) (ARS18, (GenBank Accession No. M91600) SalI/PacI YarrowiaUra3 gene (GenBank Accession No. (1493-1) AJ306421) EcoRI/BsiWIFBAIN::Δ6S::Pex20: as described for pZUF6 (1534-4251) (supra, Example19) ClaI/PacI FBAIN::YE1::Aco, comprising: (8443-1) FBAIN: FBAINpromoter (SEQ ID NO: 162) YE1: Yarrowia YE1 gene (SEQ ID NO: 64; GenBankAccession No. CAG83378) Aco: Aco3 terminator sequence from Yarrowia Aco3gene (Genbank Accession No. AJ001301)

Following transformation, transformants were grown for 2 days insynthetic MM supplemented with amino acids, followed by 4 days in HGM.The fatty acid profile of six clones containing pZUF6S and five clonescontaining pZUF6FYE1 are shown below in Table 44, based on GC analysis(as described in the General Methods). Fatty acids are identified as16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2(LA) and GLA; and the composition of each is presented as a % of thetotal fatty acids.

TABLE 44 Comparison Of Fatty Acid Composition In Yarrowia Strain Y2031Transformed With pZUF6S And pZUF6FYE1 Fatty Acid Composition (% Of TotalFatty Acids) Transformants 16:0 16:1 18:1 18:2 GLA pZUF6S #1 (control)12.9 18.2 29.6 23.5 10.7 pZUF6S #2 (control) 12.6 18.6 29.6 23.8 10.3pZUF6S #3 (control) 13.0 17.8 29.8 23.9 10.6 pZUF6S #4 (control) 13.118.9 30.1 22.3 10.3 pZUF6S #5 (control) 13.0 17.8 29.6 23.4 10.9 pZUF6S#6 (control) 12.0 18.7 30.4 23.2 10.4 Average 12.8 18.3 29.9 23.4 10.5pZUF6FYE1 #1 17.4 21.9 20.4 19.2 16.9 pZUF6FYE1 #2 16.7 22.8 21.1 19.116.1 pZUF6FYE1 #3 19.8 20.7 22.8 17.0 15.8 pZUF6FYE1 #4 16.8 22.4 23.716.1 16.8 pZUF6FYE1 #5 17.7 21.6 21.2 18.0 17.2 Average 17.7 21.9 21.917.9 16.5

GC analyses measured about 31.1% C16 (C16:0+C16:1) of total lipidsproduced in the Y2031 transformants with pZUF6S, while there was about39.6% C16 produced in the Y2031 transformants with pZUF6FYE1. The totalamount of C16 increased about 26.7% in the pZUF6FYE1 transformants, ascompared to transformants with pZUF6S. Thus, these data demonstratedthat YE1 functions as a C_(14/16) fatty acid elongase to produce C16fatty acids in Yarrowia. Additionally, there was 57% more GLA producedin the pZUF6FYE1 transformants than in pZUF6S transformants, suggestingthat the YE1 elongase could push carbon flux into the engineered pathwayto produce more final product (i.e., GLA).

Example 21 Yarrowia lipolytica CPT1 Overexpression Increases PercentPUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2067U (Example 10) that wastransformed to overexpress the Y. lipolytica CPT1 cDNA (SEQ ID NO:109).PUFAs leading to the synthesis of EPA were also increased. It iscontemplated that a Y. lipolytica host strain engineered to produce ARAvia either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8desaturase pathway could demonstrate increased ARA biosynthesis andaccumulation, if the Y. lipolytica CPT1 was similarly co-expressed(e.g., in strains Y2034, Y2047 or Y2214).

Y. lipolytica strain ATCC #20326 cDNA was prepared using the followingprocedure. Cells were grown in 200 mL YPD medium (2% Bacto-yeastextract, 3% Bactor-peptone, 2% glucose) for 1 day at 30° C. and thenpelleted by centrifugation at 3750 rpm in a Beckman GH3.8 rotor for 10min and washed twice with HGM. Washed cells were resuspended in 200 mLof HGM and allowed to grow for an additional 4 hrs at 30° C. Cells werethen harvested by centrifugation at 3750 rpm for 10 min in 4×50 mLtubes.

Total RNA was isolated using the Qiagen RNeasy total RNA Midi kit. Todisrupt the cells, harvested cells were resuspended in 4×600 μl of kitbuffer RLT (supplemented with β-mercaptoethanol, as specified by themanufacturer) and mixed with an equal volume of 0.5 mm glass beads infour 2 mL screwcap tubes. A Biospec Mini-beadbeater was used to breakthe cells for 2 min at the Homogenization setting. An additional 4×600μl buffer RLT was added. Glass beads and cell debris were removed bycentrifugation, and the supernatant was used to isolate total RNAaccording to manufacturer's protocol.

PolyA(+)RNA was isolated from the above total RNA sample using a QiagenOligotex mRNA purification kit according to the manufacturer's protocol.Isolated polyA(+) RNA was purified one additional round with the samekit to ensure the purity of mRNA sample. The final purified poly(A)+RNAhad a concentration of 30.4 ng/μl.

cDNA was generated, using the LD-PCR method specified by BD-Clontech and0.1 μg of polyA(+) RNA sample, as described in Example 13, with theexception that the PCR thermocycler conditions used for 1^(st) strandcDNA synthesis were set for 95° C. for 20 sec, followed by 20 cycles of95° C. for 5 sec and 68° C. for 6 min. The PCR product was quantitatedby agarose gel electrophoresis and ethidium bromide staining.

The Y. lipolytica CPT1 cDNA was cloned as follows. Primers CPT1-5′-NcoIand CPT1-3′-NotI (SEQ ID NOs:326 and 327) were used to amplify the Y.lipolytica ORF from the cDNA of Y. lipolytica bp PCR. The reactionmixture contained 0.5 μl of the cDNA, 0.5 μl each of the primers, 11 μlwater and 12.5 μl ExTaq premix 2×Taq PCR solution (TaKaRa Bio Inc.,Otsu, Shiga, 520-2193, Japan). Amplification was carried out as follows:initial denaturation at 94° C. for 300 sec, followed by 30 cycles ofdenaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, andelongation at 72° C. for 60 sec. A final elongation cycle at 72° C. for10 min was carried out, followed by reaction termination at 4° C. A˜1190 bp DNA fragment was obtained from the PCR reaction. It waspurified using Qiagen's PCR purification kit according to themanufacturer's protocol. The purified PCR product was digested with NcoIand NotI, and cloned into Nco I-Not I cut pZUF17 vector (SEQ ID NO:118;FIG. 8B), such that the gene was under the control of the Y. lipolyticaFBAIN promoter and the PEX20-3′ terminator region. Correct transformantswere confirmed by miniprep analysis and the resultant plasmid wasdesignated as “pYCPT1-17” (SEQ ID NO:152).

To integrate the chimeric FBAIN::CPT1::PEX20 gene into the genome ofYarrowia lipolytica, plasmid pYCPT1-ZP217 was created by digestingpYCPT1-17 with NcoI and NotI, and isolating the ˜1190 bp fragment thatcontained the CPT1 ORF. This fragment was then cloned into pZP2I7+Ura(SEQ ID NO:153) digested with NcoI and NotI. As shown in FIG. 19C,plasmid pZP2I7+Ura is a Y. lipolytica integration plasmid comprising achimeric TEF::synthetic Δ17 desaturase (codon-optimized for Y.lipolytica)::Pex20-3′ gene and a Ura3 gene, for use as a selectablemarker. Correct transformants were confirmed by miniprep analysis andthe resultant plasmid was designated as “pYCPT1-ZP217” (SEQ ID NO:154).

Y. lipolytica strain Y2067U (from Example 10) was transformed withBssHII/BbuI digested pYCPT1-ZP217 and pZUF-MOD-1 (supra, Example 14),respectively, according to the General Methods. Transformants were grownfor 2 days in synthetic MM supplemented with amino acids, followed by 4days in HGM. The fatty acid profile of two transformants containingpZUF-MOD-1 and four transformants having pYCPT1-ZP217 integrated intothe genome are shown below in the Table, based on GC analysis (asdescribed in the General Methods). Fatty acids are identified as 18:0,18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and thecomposition of each is presented as a % of the total fatty acids.

TABLE 45 Lipid Composition In Yarrowia Strain Y2067U Engineered ToOverexpress Y. lipolytica CPT1 Total Fatty Acids Strain 18:0 18:1 18:2GLA DGLA ARA ETA EPA Y2067U + pZUF-MOD-1 #1 1.3 6.9 12.0 23.1 5.7 1.13.8 13.2 Y2067U + pZUF-MOD-1 #2 1.4 6.8 12.1 22.0 5.8 1.1 3.8 13.5Y2067U + pYCPT1-ZP2I7 #1 0.6 8.0 8.2 27.4 7.1 1.6 4.1 15.7 Y2067U +pYCPT1-ZP2I7 #2 0.6 8.1 8.2 27.2 7.0 1.6 4.0 15.7 Y2067U + pYCPT1-ZP2I7#3 1.0 7.9 8.0 24.7 6.1 1.6 3.2 15.5 Y2067U + pYCPT1-ZP2I7 #4 0.6 7.18.6 25.5 6.9 1.8 4.0 16.0

As shown above, expression of the Y. lipolytica CPT1 under the controlof the strong FBAIN promoter, by genome integration, increased the % EPAfrom 13.4% in the “control” strains to 15.7-16%. Furthermore, GLA, DGLAand ARA levels also were increased.

Example 22 Sacchromyces cerevisiae ISC1 Increases Percent PUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain M4 (Example 4) that wastransformed to co-express the S. cerevisiae ISC1 gene (SEQ ID NO:111).It is contemplated that a Y. lipolytica host strain engineered toproduce ARA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9elongase/Δ8 desaturase pathway could demonstrate increased ARAbiosynthesis and accumulation, if the S. cerevisiae ISC1 was similarlyco-expressed (e.g., in strains Y2034, Y2047 or Y2214).

The S. cerevisiae ISC1 ORF was cloned into plasmid pZP2I7+Ura asfollows. First, the ORF was PCR-amplified using genomic DNA from S.cerevisiae strain S288C (Promega, Madison, Wis.) and primer pair Isc1Fand Isc1R (SEQ ID NOs:328 and 329). Primer Isc1F modified the wildtype5′ sequence of ISC1 from ‘ATGTACAA’ to ‘ATGGACAA’ in the amplified ORF,as it was necessary to incorporate a NcoI site and thereby keep ISC1 inframe. Amplification was carried out as follows: initial denaturation at94° C. for 120 sec, followed by 35 cycles of denaturation at 94° C. for30 sec, annealing at 50° C. for 30 sec and elongation at 68° C. for 120sec. A final elongation cycle at 68° C. for 10 min was carried out,followed by reaction termination at 4° C. A 1455 bp DNA fragment wasobtained from the PCR reaction for ISC1 and the PCR product size wasconfirmed by electrophoresis, using a 1% agarose gel (120 V for 30 min)and a 1 kB DNA standard ladder from Invitrogen (Carlsbad, Calif.).

The DNA was purified using a DNA Clean & Concentrator-5 kit from ZymoResearch Corporation (Orange, Calif.), per the manufacturer'sinstructions, and then digested with NcoI/NotI. The ISC1 fragment wasthen individually cloned into pZP2I7+Ura (SEQ ID NO:153; FIG. 19C)digested with NcoI and NotI. Correct transformants were confirmed by gelelectrophoresis and the resultant plasmid was designated as “pTEF::ISC1”(SEQ ID NO:155). Thus, this plasmid contained a DNA cassette comprisingthe following: 3′-PDX2, URA3, TEF::ISC1::Pex20 and a PDX2 promoterregion.

“Control” vector was prepared as follows. First, the S. cerevisiae pcl1ORF (encoding a protein involved in entry into the mitotic cell cycleand regulation of morphogenesis) was PCR amplified using genomic DNAfrom S. cerevisiae strain S288C and primer pair PcI1F and PcI1R (SEQ IDNOs:330 and 331). Amplification was carried out as described above. A861 bp DNA fragment was obtained from the PCR reaction for pcl1(confirmed by electrophoresis, supra). The DNA was purified using a DNAClean & Concentrator-5 kit and then digested with NcoI/NotI. Thefragment was then cloned into similarly digested pZP2I7+Ura. Correcttransformants were confirmed by gel electrophoresis and the resultantplasmid was designated as “pTEF::pcl1”. Plasmid pTEF::plc1 was thendigested with HincII to remove the pcl1 ORF. The remaining plasmid wasreligated, such that a linear DNA cassette comprising 3′-PDX2, URA3,TEF::Pex20 and a PDX2 promoter region resulted upon digestion withAscI/SphI.

Competent Y. lipolytica strain M4 cells (from Example 4) weretransformed with Asc1/SphI-digested pTEF::ISC1 and “control”,respectively (wherein 5 μg of each plasmid had been subject todigestion). Transformation was accomplished using the Frozen EZ YeastTransformation II kit (Zymo Research) and transformants were selected onplates comprising YNB without Amino Acids (6.7 g/L; Becton, Dickinsonand Co., Sparks, Md. [Catalog #291940]), glucose (20 g/L) and agar (20g/L). Several hundred transformant colonies were obtained. Integrationof each DNA cassette into the Yarrowia lipolytica PDX2 locus wasconfirmed by PCR using the genomic DNA from 5 independent transformantsfor ISC1.

Transformants were grown in YNB without amino acids containing 2%glucose for 2 days. The cells were harvested by centrifugation andresuspended in media comprising 100 g/L dextrose, 2 g/L MgSO₄ and 50 mMphosphate buffer at pH 6.5 for 5 additional days of growth. The cellsfrom 0.75 mL of each culture were harvested by centrifugation andanalyzed for their fatty acid composition. The fatty acid profile of 3transformants comprising the “control” vector and 5 transformantscomprising pTEF::ISC1 are shown below based on GC analysis (as describedin the General Methods). Fatty acids are identified as 16:0, 16:1, 18:0,18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and thecomposition of each is presented as a % of the total fatty acids.

TABLE 46 Lipid Composition In Yarrowia Strain M4 Engineered ToOverexpress S. cerevisiae ISC1 Total Fatty Acids Strain 16:0 16:1 18:018:1 18:2 GLA DGLA ARA ETA EPA M4 + “control” 14.7 7.2 2.1 13.5 8.7 21.88.9 0.9 4.1 9.3 M4 + pTEF::ISC1 13.5 8.5 1.7 15.6 8.1 21.3 7.5 0.7 3.910.7

Expression of the S. cerevisiae ISC1 gene improved the percent EPA from9.3% in the “control” strain to 10.7% (“M4+pTEF::ISC1”), representing a14.5% increase.

Example 23 Generation of Yarrowia lipolytica Acyltransferase Knockouts

The present Example describes the creation of single, double and tripleknockout strains of Yarrowia lipolytica that were disrupted in eitherPDAT, DGAT2, DGAT1, PDAT and DGAT2, PDAT and DGAT1, DGAT1 and DGAT2, orPDAT, DGAT1 and DGAT2 genes. Disruption of the gene(s) in each of theknock-out strains was confirmed and analysis of each of the disruptionson fatty acid content and composition was determined by GC analysis oftotal lipids in Example 24.

Targeted Disruption of the Yarrowia lipolytica DGAT2 Gene

Targeted disruption of the DGAT2 gene in Y. lipolytica ATCC #90812 wascarried out by homologous recombination-mediated replacement of theendogenous DGAT2 gene with a targeting cassette designated as plasmidpY21 DGAT2. pY21 DGAT2 was derived from plasmid pY20 (FIG. 19D; SEQ IDNO:156). Specifically, pY21 DGAT2 was created by inserting a 570 bp HindIII/Eco RI fragment into similarly linearized pY20. The 570 bp DNAfragment contained (in 5′ to 3′ orientation): 3′ homologous sequencefrom position +1090 to +1464 (of the coding sequence (ORF) in SEQ IDNO:89), a Bgl II restriction site and 5′ homologous sequence fromposition +906 to +1089 (of the coding sequence (ORF) shown in SEQ IDNO:89). The fragment was prepared by PCR amplification using two pairsof PCR primers, P95 and P96 (SEQ ID NOs:332 and 333), and P97 and P98(SEQ ID NOs:334 and 335), respectively.

pY21 DGAT2 was linearized by Bgl II restriction digestion andtransformed into mid-log phase Y. lipolytica ATCC #90812 cells,according to the General Methods. The cells were plated onto YPDhygromycin selection plates and maintained at 30° C. for 2 to 3 days.

Fourteen Y. lipolytica ATCC #90812 hygromycin-resistant colonies wereisolated and screened for targeted disruption by PCR. One set of PCRprimers (P115 and P116 [SEQ ID NOs:336 and 337]) was designed to amplifya specific junction fragment following homologous recombination. Anotherpair of PCR primers (P115 and P112 [SEQ ID NO:338]) was designed todetect the native gene.

Two of the 14 hygromycin-resistant colonies of ATCC #90812 strains werepositive for the junction fragment and negative for the native fragment.Thus, targeted integration was confirmed in these 2 strains, one ofwhich was designated as “S-D2”.

Targeted Disruption of the Yarrowia lipolytica PDAT Gene

Targeted disruption of the PDAT gene in Y. lipolytica ATCC #90812 wascarried out by homologous recombination-mediated replacement of theendogenous PDAT gene with a targeting cassette designated as pLV13 (FIG.19E; SEQ ID NO:157). pLV13 was derived from plasmid pY20 (FIG. 19D; SEQID NO:156). Specifically, the hygromycin resistant gene of pY20 wasreplaced with the Yarrowia Ura3 gene to create plasmid pLV5. Then, pLV13was created by inserting a 992 bp Bam HI/Eco RI fragment into similarlylinearized pLV5. The 992 bp DNA fragment contained (in 5′ to 3′orientation): 3′ homologous sequence from position +877 to +1371 (of thecoding sequence (ORF) in SEQ ID NO:76), a Bgl II restriction site and 5′homologous sequence from position +390 to +876 (of the coding sequence(ORF) in SEQ ID NO:76). The fragment was prepared by PCR amplificationusing PCR primers P39 and P41 (SEQ ID NOs:339 and 340) and P40 and P42(SEQ ID NOs:341 and 342), respectively.

pLV13 was linearized by Bgl II restriction digestion and was transformedinto mid-log phase Y. lipolytica ATCC #90812 cells, according to theGeneral Methods. The cells were plated onto Bio101 DOB/CSM-Ura selectionplates and maintained at 30° C. for 2 to 3 days.

Ten Y. lipolytica ATCC #90812 colonies were isolated and screened fortargeted disruption by PCR. One set of PCR primers (P51 and P52 [SEQ IDNOs:343 and 344]) was designed to amplify the targeting cassette.Another set of PCR primers (P37 and P38 [SEQ ID NOs:345 and 346]) wasdesigned to detect the native gene. Ten of the ten strains were positivefor the junction fragment and 3 of the 10 strains were negative for thenative fragment, thus confirming successful targeted integration inthese 3 strains. One of these strains was designated as “S-P”.

Targeted Disruption of the Yarrowia lipolytica DGAT1 Gene

The full-length YI DGAT1 ORF was cloned by PCR using degenerate PCRprimers P201 and P203 (SEQ ID NOs:347 and 348, respectively) and Y.lipolytica ATCC #76982 genomic DNA as template. The degenerate primerswere required, since the nucleotide sequence encoding YI DGAT1 was notknown.

The PCR was carried out in a RoboCycler Gradient 40 PCR machine, withamplification carried out as follows: initial denaturation at 95° C. for1 min, followed by 30 cycles of denaturation at 95° C. for 30 sec,annealing at 55° C. for 1 min, and elongation at 72° C. for 1 min. Afinal elongation cycle at 72° C. for 10 min was carried out, followed byreaction termination at 4° C. The expected PCR product (ca. 1.6 kB) wasdetected by agarose gel electrophoresis, isolated, purified, cloned intothe TOPO® cloning vector (Invitrogen), and partially sequenced toconfirm its identity.

Targeted disruption of the putative DGAT1 gene in Y. lipolytica ATCC#90812 was carried out by homologous recombination-mediated replacementof the endogenous DGAT1 gene with a targeting cassette (using themethodology described above for DGAT2). Specifically, the 1.6 kBisolated YI DGAT1 ORF (SEQ ID NO:81) was used as a PCR template moleculeto construct a YI DGAT1 targeting cassette consisting of: 5′ homologousYI DGAT1 sequence (amplified with primers P214 and P215 (SEQ ID NOs:349and 350)), the Yarrowia Leucine 2 (Leu2; GenBank Accession No. AAA35244)gene, and 3′ homologous YI DGAT1 sequence (amplified with primers P216and P217 (SEQ ID NOs:351 and 352)). Following amplification of eachindividual portion of the targeting cassette with Pfu Ultra polymerase(Stratagene, Catalog #600630) and the thermocycler conditions describedabove, each fragment was purified. The three correct-sized, purifiedfragments were mixed together as template molecules for a second PCRreaction using PCR primers P214 and P219 (SEQ ID NO:353) to obtain theYI DGAT1 disruption cassette.

The targeting cassette was gel purified and used to transform mid-logphase wildtype Y. lipolytica (ATCC #90812). Transformation was performedas described in the General Methods. Transformants were plated ontoBio101 DOB/CSM-Leu selection plates and maintained at 30° C. for 2 to 3days. Several leucine prototrophs were screened by PCR to confirm thetargeted DGAT1 disruption. Specifically, one set of PCR primers (P226and P227 [SEQ ID NOs:354 and 355]) was designed to amplify a junctionbetween the disruption cassette and native target gene. Another set ofPCR primers (P214 and P217 [SEQ ID NOs:349 and 352]) was designed todetect the native gene.

All of the leucine prototroph colonies were positive for the junctionfragment and negative for the native fragment. Thus, targetedintegration was confirmed in these strains, one of which was designatedas “S-D1”.

Creation of Yarrowia lipolytica Double and Triple Knockout StrainsContaining Disruptions in PDAT and/or DGAT2 and/or DGAT1 Genes

The Y. lipolytica ATCC #90812 hygromycin-resistant “S-D2” mutant(containing the DGAT2 disruption) was transformed with plasmid pLV13(containing the PDAT disruption) and transformants were screened by PCR,as described for the single PDAT disruption. Two of twelve transformantswere confirmed to be disrupted in both the DGAT2 and PDAT genes. One ofthese strains was designated as “S-D2-P”.

Similarly, strains with double knockouts in DGAT1 and PDAT (“S-D1-P”),in DGAT2 and DGAT1 (“S-D2-D1”), and triple knockouts in DGAT2, DGAT1 andPDAT (“S-D2-D1-P”) were made.

Example 24 Yarrowia lipolytica Acyltransferase Knockouts Decrease LipidContent and Increase Percent PUFAs

The present Example analyzes the affect of single and/or double and/ortriple acyltransferase knockouts in wildtype Yarrowia lipolytica andstrains of Y. lipolytica that had been previously engineered to produceEPA, as measured by changes in fatty acid content and composition. It iscontemplated that a Y. lipolytica host strain engineered to produce ARAvia either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8desaturase pathway could demonstrate increased ARA biosynthesis andaccumulation, if similar manipulations to the host's nativeacyltransferases were created (e.g., within strains Y2034, Y2047 orY2214).

TAG Content is Decreased in Y. Lipolytica ATCC #90812 withAcyltransferase Disruptions

First, TAG content was compared in wildtype and mutant Y. lipolyticaATCC #90812 containing: (1) single disruptions in PDAT, DGAT2 and DGAT1;(2) double disruptions in PDAT and DGAT2, DGAT1 and PDAT, and DGAT1 andDGAT2; and (3) triple disruptions in PDAT, DGAT2 and DGAT1.

Specifically, one loopful of cells from plates containing wildtype andmutant Y. lipolytica ATCC #90812 (i.e., strains S-D1, S-D2, S-P,S-D1-D2, S-D1-P, S-D2-P, and S-D1-D2-P) were each individuallyinoculated into 3 mL YPD medium and grown overnight on a shaker (300rpm) at 30° C. The cells were harvested and washed once in 0.9% NaCl andresuspended in 50 mL of HGM. Cells were then grown on a shaker for 48hrs. Cells were washed in water and the cell pellet was lyophilized.Twenty (20) mg of dry cell weight was used for total fatty acid by GCanalysis and the oil fraction following TLC (infra) and GC analysis.

The methodology used for TLC is described below in the following fivesteps: (1) The internal standard of 15:0 fatty acid (10 μl of 10 mg/mL)was added to 2 to 3 mg dry cell mass, followed by extraction of thetotal lipid using a methanol/chloroform method. (2) Extracted lipid (50μl) was blotted across a light pencil line drawn approximately 1 inchfrom the bottom of a 5×20 cm silica gel 60 plate, using 25-50 μlmicropipettes. (3) The TLC plate was then dried under N₂ and wasinserted into a tank containing about ˜100 mL 80:20:1 hexane:ethylether:acetic acid solvent. (4) After separation of bands, a vapor ofiodine was blown over one side of the plate to identify the bands. Thispermitted samples on the other side of the plate to be scraped using arazor blade for further analysis. (5) Basic transesterification of thescraped samples and GC analysis was performed, as described in theGeneral Methods.

GC results are shown below in Table 47. Cultures are described as the“S” strain (wildtype), “S-P” (PDAT knockout), “S-D1” (DGAT1 knockout),“S-D2” (DGAT2 knockout), “S-D1-D2” (DGAT1 and DGAT2 knockout), “S-P-D1”(PDAT and DGAT1 knockout), “S-P-D2” (PDAT and DGAT2 knockout) and“S-P-D1-D2” (PDAT, DGAT1 and DGAT2 knockout). Abbreviations utilizedare: “WT”=wildtype; “FAs”=fatty acids; “dcw”=dry cell weight; and, “FAs% dcw, % WT”=FAs % relative to the % in wildtype, wherein the “S” strainis wildtype.

TABLE 47 Lipid Content In Yarrowia ATCC #90812 Strains With Single,Double, Or Triple Disruptions In PDAT, DGAT2 And DGAT1 Total Fatty AcidsTAG Fraction FAs % FAs % Residual FAs, FAs % dcw, % FAs, FAs % dcw, %Strain DAG AT dcw, mg μg dcw WT μg dcw WT S D1, D2, P 32.0 797 15.9 100697 13.9 100 S-D1 D2, P 78.8 723 13.6 86 617 11.6 83 S-D2 D1, P 37.5 3296.4 40 227 4.4 32 S-P D1, D2 28.8 318 6.0 38 212 4.0 29 S-D1-D2 P 64.6219 4.1 26 113 2.1 15 S-D1-P D2 76.2 778 13.4 84 662 11.4 82 S-D2-P D131.2 228 4.3 27 122 2.3 17 S-D1-D2-P None 52.2 139 2.4 15 25 0.4 3

The results in Table 47 indicate the relative contribution of the threeDAG ATs to oil biosynthesis. DGAT2 contributes the most, while PDAT andDGAT1 contribute equally but less than DGAT2. The residual oil contentca. 3% in the triple knockout strain may be the contribution of Yarrowialipolytica's acyl-CoA:sterol-acyltransferase enzyme, encoded by ARE2(SEQ ID NOs:78 and 79).

TAG Content is Decreased and Percent EPA is Increased in Yarrowialipolytica Strain EU with a Disrupted DGAT2 Gene

After examining the affect of various acyltransferase knockouts inwildtype Y. lipolytica ATCC #90812 (supra), TAG content and fatty acidcomposition was then studied in DGAT2 knockout strains of the EU strain(i.e., engineered to produce 10% EPA; see Example 10).

Specifically, the DGAT2 gene in strain EU was disrupted as described forthe S strain (ATCC #90812) in Example 23. The DGAT2-disrupted strain wasdesignated EU-D2. EU and EU-D2 strains were harvested and analyzedfollowing growth according to two different conditions. In the conditionreferred to in the Table below as “3 mL”, cells were grown for 1 day in3 mL MM medium, washed and then grown for 3 days in 3 mL HGM.Alternatively, in the condition referred to in the Table below as “51mL”, cells were grown for 1 day in 51 mL MM medium, washed and thengrown for 3 days in 51 mL HGM. The fatty acid compositions ofphosphatidylcholine (PC), phosphatidyletanolamine (PE), andtriacylglycerol (TAG or oil) were determined in the extracts of 51 mLcultures following TLC separation (“Fraction”).

GC results are shown below in Table 48. Cultures are described as the“EU” strain (wildtype) and the “EU-D2” strain (DGAT2 knockout). Fattyacids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid), 18:2 (LA),GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented asa % of the total fatty acids.

TABLE 48 Lipid Content And Composition In Yarrowia Strain EU WithDisruption In DGAT2 Strain & TFAs % % % % % % % % % % % Growth Fractiondcw 16:0 16:1 18:0 18:1 18:2 GLA DGLA ARA ETA EPA EU, Total 19 10 2 1612 19 6 0 3 10 3 mL EU- Total 17 10 1 6 7 24 5 0 6 19 D2, 3 mL EU, Total37 18 11 3 19 31 5 1 1 4 51 mL PC 2 12 9 1 8 43 7 3 5 4 PE 1 24 14 0 1437 5 0 0 1 TAG 34 18 12 3 21 29 5 1 1 4 EU- Total 18 18 8 1 5 7 25 5 520 D2, 51 mL PC 1 18 6 1 2 4 26 5 11 22 PE 1 25 7 0 2 5 14 2 3 8 TAG 1516 9 1 6 5 26 6 5 21

The results show that the DGAT2 knockout resulted in doubling of the %EPA (of total fatty acids) and halving of the lipid content (as % dcw).Furthermore, almost all of the changes observed in the lipid content aredue to changes in the TAG fraction. The lower than expected % EPA in the51 mL culture of strain EU is likely due to instability.

TAG Content is Decreased and Percent EPA is Increased in Yarrowialipolytica Strain MU with Disrupted Acyltransferase Genes

Finally, based on the increased % EPA and reduced lipid contentresulting from a single DGAT2 knockout in strain EU-D2, TAG content andfatty acid composition was then studied in various acyltransferaseknockout strains of strain MU (engineered to produce 14% EPA; seeExample 12). Specifically, single disruptions in PDAT, DGAT2 and DGAT1and double disruptions in PDAT and DGAT2 were created in strain MU.Lipid content and composition was compared in each of these strains,following growth in 4 different growth conditions.

More specifically, single disruptions in PDAT, DGAT2, DGAT1 were createdin strain MU, using the methodology described in Example 23 (with theexception that selection for the DGAT1 disruption relied on the URA3gene). This resulted in single knockout strains identified as “MU-D1”(disrupted in DGAT1), “MU-D2” (disrupted in DGAT2), and “MU-P”(disrupted in PDAT). Individual knockout strains were confirmed by PCR.Additionally, the MU-D2 strain was disrupted for the PDAT gene by thesame method and the disruption confirmed by PCR. The resulting doubleknockout strain was designated “MU-D2-P”.

The MU-D1, MU-D2, MU-P, and M-D2-P knockout strains were analyzed todetermine each knockout's effect on lipid content and composition, asdescribed below. Furthermore, the growth conditions promoting oleaginywere also explored to determine their effect on total lipid content.Thus, in total, four different experiments were conducted, identified as“Experiment A”, “Experiment B”, “Experiment C” and “Experiment E”.Specifically, three loops of cells from plates containing each strainabove was inoculated into MMU medium [3 mL for Experiments B and C; and50 mL for Experiments A and E] and grown in a shaker at 30° C. for 24hrs (for Experiments A, B and C) or 48 hrs (for Experiment E). Cellswere harvested, washed once in HGM, resuspended in either HGM medium (50mL for Experiments A and E; and 3 mL for Experiment B) or HGM mediumwith uracil (“HGMU”) (3 mL for Experiment C) and cultured as above for 4days. One aliquot (1 mL) was used for lipid analysis by GC as describedaccording to the General Methods, while a second aliquot was used fordetermining the culture OD at 600 nm. The remaining culture inExperiments A and E was harvested, washed once in water, and lyophilizedfor dry cell weight (dcw) determination. In contrast, the dcw inExperiments B and C were determined from their OD₆₀₀ using the equationshowing their relationship. The fatty acid compositions of each of thedifferent strains in Experiments A, B, C and E was also determined.

The results are shown in Table 49 below. Cultures are described as the“MU” strain (the parent EPA producing strain), “MU-P” (PDAT knockout),“MU-D1” (DGAT1 knockout), “MU-D2” (DGAT2 knockout) and “MU-D2-P” (DGAT2and PDAT knockouts). Abbreviations utilized are: “WT”=wildtype (i.e.,MU); “OD”=optical density; “dcw”=dry cell weight; “TFAs”=total fattyacids; and, “TFAs % dcw, % WT”=TFAs % dcw relative to the wild type(“MU”) strain. Fatty acids are identified as 16:0, 16:1, 18:0, 18:1(oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and thecomposition of each is presented as a % of the total fatty acids.

TABLE 49 Lipid Content And Composition In Yarrowia Strain MU WithVarious Acyltransferase Disruptions 1^(st) Phase 2^(nd) Phase TFAs %Residual Growth Growth dcw TFAs TFAs dcw, Expt Strain DAG AT ConditionCondition OD (mg) (μg) % dcw % WT A MU D1, D2, P 1 day, 4 days, 4.0 91374 20.1 100 A MU-D2 D1, P 50 mL 50 mL 3.1 75 160 10.4 52 A MU-D1 D2, PMMU HGM 4.3 104 217 10.2 51 A MU-P D1, D2 4.4 100 238 11.7 58 B MU D1,D2, P 1 day, 4 days, 5.9 118 581 24.1 100 B MU-D2 D1, P 3 mL 3 mL 4.6102 248 11.9 50 B MU-D1 D2, P MMU HGM 6.1 120 369 15.0 62 B MU-P D1, D26.4 124 443 17.5 72 C MU D1, D2, P 1 day, 4 days, 6.8 129 522 19.9 100 CMU-D2 D1, P 3 mL 3 mL 5.6 115 239 10.2 51 C MU-D1 D2, P MMU HGMU 6.9 129395 15.0 75 C MU-P D1, D2 7.1 131 448 16.8 84 E MU D1, D2, P 2 days, 4days, 4.6 89 314 17.3 100 E MU-D2 D1, P 50 mL 50 mL 2.8 62 109 8.5 49 EMU-P D2, P MM HGM 5.0 99 232 11.5 66 E MU-D2-P D1 4.2 98 98 4.9 28 % % %% % % % % % % Expt Strain 16:0 16:1 18:0 18:1 18:2 GLA DGLA ARA ETA EPAA MU 17 10 2 18 10 22 7 1 3 9.7 A MU-D2 16 12 0 8 9 23 7 0 8 17.4 AMU-D1 15 10 2 11 10 22 7 0 7 17.4 A MU-P 16 9 2 11 7 24 7 1 6 17.5 B MU17 9 3 18 10 22 8 1 3 9.1 B MU-D2 16 10 0 7 10 24 7 1 7 17.8 B MU-D1 189 3 14 11 20 7 1 5 12.0 B MU-P 15 8 3 16 10 25 6 1 4 11.9 C MU 16 10 213 11 21 10 1 4 12.6 C MU-D2 17 9 1 6 11 21 8 1 7 18.9 C MU-D1 15 9 2 1212 20 10 1 5 13.5 C MU-P 17 8 3 14 11 20 10 1 4 11.3 E MU 16 12 2 18 922 7 1 4 11.2 E MU-D2 14 12 1 6 8 25 6 0 7 20.0 E MU-P 16 10 2 14 7 24 71 5 15.8 E MU-D2-P 18 10 0 7 12 20 5 0 6 22.5

The data showed that the lipid content within the transformed cellsvaried according to the growth conditions. Furthermore, the contributionof each acyltransferase on lipid content also varied. Specifically, inExperiments B, C and E, DGAT2 contributed more to oil biosynthesis thaneither PDAT or DGAT1. In contrast, as demonstrated in Experiment A, asingle knockout in DGAT2, DGAT1 and PDAT resulted in approximatelyequivalent losses in lipid content (i.e., 48%, 49% and 42% loss,respectively [see “TFAs % dcw, % WT”]).

With respect to fatty acid composition, the data shows that knockout ofeach individual DAG AT gene resulted in lowered oil content andincreased % EPA. For example, the DGAT2 knockout resulted in about halfthe lipid content and ca. double the % EPA in total fatty acids (similarto the results observed in strain EU-D2, supra). Knockout of both DAGAT2and PDAT resulted in the least oil and the most % EPA.

On the basis of the results reported herein, it is contemplated thatdisruption of the native DGAT2 and/or DGAT1 and/or PDAT is a usefulmeans to substantially increase the % PUFAs in a strain of Yarrowialipolytica engineered to produce high concentrations of PUFAs, includingARA (e.g., strains Y2034, Y2047, Y2214). In fact, a disruption of thenative DGAT2 gene in strain Y2214 resulted in a 1.7 fold increase in thepercent ARA (data not shown).

1-36. (canceled)
 37. A microbial oil comprising: a) at least 14%arachidonic acid as a weight percent of the total fatty acids of themicrobial oil, and b) less than 5% stearic acid as a weight percent ofthe total fatty acids of the microbial oil; wherein said microbial oilis devoid of gamma-linolenic acid.
 38. The microbial oil of claim 37,wherein the oil comprises at least about 25% arachidonic acid as aweight percent of the total fatty acids of the microbial oil.
 39. Themicrobial oil of claim 37, wherein the oil is devoid of docosahexaenoicacid.
 40. The microbial oil of claim 38, wherein the oil is devoid ofdocosahexaenoic acid.
 41. The microbial oil of claim 37, wherein the oilcomprises less than 2% stearic acid as a weight percent of the totalfatty acids of the microbial oil.
 42. The microbial oil of claim 41,wherein the oil is devoid of stearic acid.